Long-term culture of adult progenitor cells in 3D is a recently emerging technology that inhabits the space between 2D cell lines and organ slice culture.Adaptations to defined media components in the wake of advances in ES and iPS cell culture has led to the identification of conditions that maintained intestinal cell progenitors in culture. These conditions retain cellular heterogeneity of the normal or tumour tissue, and the cultures have been shown to be genetically stable, such that substantial biobanks are being created from patient derived material. This coupled with advances in analytical tools has generated a field, characterized by the term “organoid culture”, that has huge potential for advancing drug discovery, regenerative medicine, and furthering the understanding of fundamental intestinal biology.In this review, we describe the approaches available for the long-term culture of intestinal cells from normal and diseased tissue, the current challenges, and how the technology is likely to develop further. Lgr5 as an ICP marker and publication of a 3D culture technique which allowed single murine intestinal stem cells to be grown into organoids that contained protruding crypt structures with all the cell lineages that comprise the small intestinal crypt in vivo [Lgr5 driven GFP reporter [Bmi1 [2 (that promoted organoid integrity through blocking anoikis and promoting proliferation), and Nicotinamide (a vitamin shown to inhibit differentiation) [In 2007, after decades of research, the intestinal cell progenitors (ICPs) that are responsible for regenerating the surface of the normal intestine every 2–7 days were identified and shown to reside within crypt bases of the intestinal epithelium . Up to t in vivo , 5•. The in vivo ) in a mereporter  has enabreporter  along wier [Bmi1 , and inder [Bmi1 . This wotiation) , 12.This review discusses the progress made over the last 3 years in using organoid culture of tissue-derived ICPs. Related developments in which intestinal cultures are generated by the directed differentiation of embryonic or induced pluripotent stem cells are described and reviewed elsewhere –16. WithICP-generated 3D organoids retain in vivo cell-to-cell contacts, mass transport properties, mechanical properties, and metabolic profiles, whilst incorporating many cell types, modelling cell proliferation/differentiation, combined with long-term genomic stability • and genEx vivo ICP culture has been highly relevant in CRC, where the ISC has been identified as the cell of origin . PreviouThere are a number of paths to modelling CRC based upon the culture of ICPs, perhaps the most direct involving the collection of patient tumour material and the preparation of stable long-term cultures. This has led to the development of CRC organoid collections (or biobanks) which have the aim of representing the diversity of CRC disease, infection, and drug responses that exists within the patient population •, 26, 27Troy [Brg1 [Cdx2 [Kcnq [Prox1 [Fzd7 [Yap [FBXW7-mutated CRC cells to certain types of chemotherapy (e.g. Fluorouracil (5-FU)) is due to an inhibition of terminal differentiation indicating the that they could be overcome by using differentiating therapies [Elp3 plays a key role in the radio-resistance of the Lgr5+Dclk1+ malignant ISC due to its promotion of Sox9 translation [The second major source of malignant ICP cultures is from genetically characterized GEMMs. Despite the drawbacks previously mentioned, they are still invaluable tools for research and can be combined with organoid culture to great effect. This was epitomized by the work from the group of Doug Winton, who used ICPs from GEMMs to generate organoids that could contain a singly mutated ICP to understand the altered crypt dynamics and clonal advantage induced by the most common genetic alterations pervading CRC biology  . Indeed Troy , Brg1 [3oy [Brg1 , Cdx2 [3g1 [Cdx2 , Kcnq [3x2 [Kcnq , Prox1 [q [Prox1 , Fzd7 [3x1 [Fzd7 , Yap [36herapies . ICP culherapies . It has nslation . There aApcmin/+ mice noting an aberrant cyst-like sphere morphology induced by a constitutively activated Wnt pathway that was responsible for increasing both self-renewal and growth while reducing differentiation. This observation combined with deletion studies elegantly described the engagement of the Wnt, Notch, and Myb transcriptional pathways in intestinal tumourigenesis and further highlighted the Wnt pathway as a therapeutic target in CRC. It seems likely that these last techniques will slowly lead us away from our current reliance on GEMMs.A third approach capable of utilizing both of the above CRC culture derivations is through the exploitation of current DNA manipulation technologies to manipulate normal mouse and human ICPs into cancer ICPs that reflect human CRC. Using shRNA to target APC, P53, and PTEN, Onuma et al. have used lentiviral vectors in normal ICPs that has enabled them to generate recapitulated intestinal tumour organoids without generating gene-modified mice . SimilarThis section summarizes in turn, personalized medicine, drug discovery, and regenerative medicine, the three main areas of clinical application for long-term ICP cultures.One of the greatest potential benefits of ICP cultures is to deliver a personalized medicine paradigm, where much like a biomedical service such as microbiology or virobiology a small sample is supplied and then cultured for rapid analysis. Where cultures act as ‘avatars’ of patients in the dish, an emerging application recently summarized by  allows cID1 [Diverse collections of patient samples reflecting population disease profiles may power future drug discovery programmes, wherein organoids representative of specific disease subtypes would be tested against panels of compounds in drug titration assays in order to determine their potential efficacy. Key to shifting the current 2D culture led paradigm will be the generation of defined batches of organoids through scalable processes for commercial drug discovery programmes. Already the use of ICP culture is currently expanding knowledge of the role of individual genes in response to injury , and cheID1 .Increasing our understanding of the influence of diet on the ISC using ex vivo culture is identifying the mechanisms for understanding the cause of disease resistance and simultaneously opening up potential avenues to be exploited for regenerative medicine. Potentially, the relationship between a high fat diet , and susceptibility to CRC could be exploited to aid regeneration following intestinal injury . FurtherHowever, for organoids to achieve their clinical potential, biobanks of intestinal disease (i.e. CRC) would ideally (1) contain the tumour, blood (germline DNA), and early passage organoid set for DNA/expression analysis, which would allow the checking for a faithful recapitulation of the tumour by the organoid culture, alongside the identification of mutations from polymorphisms, (2) the ability to supply/maintain organoids in sufficient quantities and under standardized conditions to facilitate drug titration assays, which will require the development of bioreactors capable of standardizing growth and assay material, and (3) a sample collection reflecting a broad genetic diversity enabling toxicity studies where the range of likely toxic responses can be monitored.Increasingly, the literature demonstrates a proliferation of the use of, and the number of, applications of ICP models Table . Indeed,Aside from standardization of existing models, ongoing elaborations of ICP niche culture are being pursued in efforts to draw models still closer to recapitulating the organ in a dish Table . There aThe use of ICP culture and technology to alter gene behaviour can elicit a myriad of cellular biology; thus, there is a requirement for techniques that can be used to analyse and quantify relevant parameters within complex 3D cellular environments . This seA change from 2D to 3D biology represents a sizable image analysis challenge. Standard light microscopy image analysis (often achieved using whole well or plate scanning imaging apparatus) is limited to 2D images, offering relatively few extra parameters , although they do facilitate non-destructive serial measurements. In order to exploit the readouts of commonly used immunohistochemistry and immunofluorescence methods , microscopic imaging platforms, such as wide field and confocal high content microscopes, are being used to analyse organoid cultures in 3D. Software analysis pipelines that are capable of analysing hundreds of 3D morphometric parameters and readouts can be used to identify subtle biological effects of media conditions and drug treatments . These wThe ability to isolate and examine single cells within an organoid is an important feature of ICP culture. As the next-generation single-cell gene profiling becomes increasingly available in research labs, further progress will be made in understanding intestinal biology and disease. Existing next-generation sequencing technology has already been adapted and exploited to identify new intestinal cell types within organoids based on single cell messenger RNA sequencing . This wiFor the current rate of progress in the use of organoids to be maintained, it will be important that the development of technology, for obtaining and interpreting the vast quantities of information that can be gained from long-term ICP cultures, does not lag far behind.PPARδ-dependent link to an increase in stemness within the intestinal niche that predisposes to CRC. Although these reductionist approaches are yielding greater insight into ICP, ultimately there will need to be greater efforts made to bring ex vivo culture techniques closer to the in vivo environment. The challenge for the future is to develop the tools for ICP culture to the point where human intestine can be recapitulated in the laboratory, such as elaborate co-culturing systems involving microfluidic linked culture vessels mimicking multi-compartment and even multi-tissue interactions. This would enable the exploration of the interactions between the full range of factors  that impact on the ISC and the roles they play in promoting, preventing, initiating, and driving intestinal diseases.The complexity of the intestinal niche necessitates further elaboration of culture model systems that will likely include gut microbiota and diet research. Current models do not include stroma, elements of the immune system, a disease-specific ECM, or gut bacteria (the latter applicable to drug development for infection models). The ability of ICPs to faithfully maintain physiological relevance over time is vitally important for their use as research tools. However, replicating the environment in the human large intestine is crucial to our understanding how CRC develops. Culture conditions that manipulate the signalling pathways essential for ISC function are continually being identified and refined to better replicate and understand the ISC niche , 76. Rec


Lignin is one of the main components of plant cell wall and it is a natural phenolic polymer with high molecular weight, complex composition and structure. Lignin biosynthesis extensively contributes to plant growth, tissue/organ development, lodging resistance and the responses to a variety of biotic and abiotic stresses. In the present review, we systematically introduce the biosynthesis of lignin and its regulation by genetic modification and summarize the main biological functions of lignin in plants and their applications. We hope this review will give an in-depth understanding of the important roles of lignin biosynthesis in various plants’ biological processes and provide a theoretical basis for the genetic improvement of lignin content and composition in energy plants and crops. It is the second most profuse biopolymers that accounts for 30% of the organic carbon content in biosphere . Lignin ell wall ,3,4,5. Iell wall ,10,11,12ll walls .Lignin and its related metabolism play important roles in the growth and development of plants. As a complex phenolic polymer, lignin enhances plant cell wall rigidity, hydrophobic properties and promotes minerals transport through the vascular bundles in plant . In addiAs one of the important components of plant cell wall, lignin is of great significance to plant growth and environmental adaptability. In addition, lignin itself can also be used as a resource for the field of energy or pharmaceutical industry. The study of lignin biosynthesis and its function will have a significant impact on industrial, agricultural production and other human activities. Therefore, we briefly summarize the biosynthesis, function and application of lignin to provide a mini review of the research on lignin.Arabidopsis thaliana quadruple mutant  of PAL genes, decreased by 20–25% as compared with the wild type, while the quadruple mutant also displayed reduced levels of salicylic acid and increased susceptibility to pathogen [Os4CL3 gene expression significantly reduced the lignin content and height of rice plants. Suppression of the mRNA level of 4CL gene in Pinus radiata reduced the lignin content of transgenic plants by 36–50%, which mainly due to depletion of guaiacyl lignin and resulted in a dwarfed phenotype of plant [Arabidopsis thaliana ccc triple mutant (cad c cad d ccr1), i.e., lignin content was reduced by 50% as compared with the wild type and was accompanied by the male-sterile phenotype [CCR gene is considered to be an effective strategy for the production of low-lignin C4 Paspalumdilatatum [p-coumarate 3-hydroxylase (C3H) and caffeate/5-hydroxyconiferaldehyde O-methyltransferase (COMT) are involved in the synthesis of sinapyl alcohol (S unit) and coniferyl alcohol (G unit). Interference of the expression of CCR1 and COMT1 genes significantly altered lignin content and composition in ryegrass and enhanced digestibility without significant negative effects on either plant fitness or biomass production [Medicago sativa, expressing an HCT antisense construct, led to a significant decrease in lignin content and an obvious change in the lignin composition, while exhibited obvious stunting, decreased biomass and delayed flowering. Vanholme et al. [Populus nigra hct1 mutant had a modified H lignin disposition. Inhibition of C3H expression in poplars also reduced the lignin content of poplar and changed its S/G ratio [O-methyltransferase MOMT4 gene resulted in a 24% reduction in lignin content in Arabidopsis cells and increased saccharification yields of transgenic plants. Ferulate 5-hydroxylase (F5H) is one of key enzymes that regulate S/G lignin composition in plants, recent study has shown that the heterologous expression of angiosperm F5H induced ectopic productions of S lignin in the gymnosperm cell walls [OsCAld5H1 increased the content of S units, while down-regulation of it enhanced the production of G lignin [Arabidopsis thaliana, Medicago truncatula and hybrid poplar [Sorghum bicolor (SbCOMT) can methylate the tricin precursors (luteolin and selgin) and be involved in the biosynthesis of lignin-linked tricin and S lignin units in sorghum.Lignin content, composition and structure can be altered using genetic modification. The effects of single or multiple lignin biosynthesis gene expression on phenotypic traits of transgenic plants can be directly observed and identified . In the pathogen . Gui et pathogen  found thof plant . Cinnamohenotype . Recentlilatatum . Hydroxyoduction . Shadle oduction  showed te et al.  found th/G ratio . Zhang e/G ratio  reportedll walls . In riceG lignin . Recentld poplar ,34,35. Id poplar . Eudes eArabidopsis AtABCG29 knockout mutants exhibited less lignin content and more sensitivity to p-coumaryl alcohol, due to p-coumaryl alcohol transport activity of AtABCG29 protein [AtPrx double mutants  had lower lignin content than single mutants but did not seriously affect the growth. Recently, a new class III peroxidase PRX17 (At2g22420) in Arabidopsis thaliana was reported to participate in lignin biosynthesis and be regulated by a MADS-box transcription factor AGL15 [laccase in lignin biosynthesis is relatively poorly studied in the past years. Wang et al. [Arabidopsis laccase double mutants, lac4-1 lac17 and lac4-2 lac17, decreased by 20% and 40%, respectively. Surprisingly, lignin deposition in roots has almost completely disappeared in the Arabidopsis lac11 lac4 lac17 triple mutant [Brachypodium distachyon, which lead to a modification on lignin content and composition [After the synthesis of monomers in the cytoplasm, they are transported across the cell membrane and then polymerized in the cell wall . Arabido protein ,46. Over protein . Shigeto protein  found thor AGL15 . Compareg et al.  confirmeposition .Arabidopsis thaliana MYB58 and MYB63 transcription factors activated the expression of lignin biosynthesis-related genes and promoted the lignification of cells. While the other transcription factors, such as EgMYB1 from eucalyptus, MusaMYB31 from Musa inhibited the expression of genes related to lignin biosynthesis and negatively regulated lignin accumulation [Arabidopsis were able to control lignin biosynthesis by regulating the entire cell wall synthesis-related genes [Medicago sativa L. [O-methyl transferase gene CCoAOMT1, switchgrass UDP-Arabinomutase gene, Betula platyphylla MADS-box gene BpMADS12 and Eriobotrya japonica heat shock factors EjHSF3 [Studies have shown that phenylpropanoid metabolism and lignin-specific synthesis can be regulated by transcription factors. The most widely studied transcription factor for lignin biosynthesis is the MYB  family. It has been found that overexpression of mulation ,53,54. Iativa L. . In the s EjHSF3 ,43,56,57Arabidopsis thaliana mutant seed coat significantly decreased the seed germination rate, in comparison to wild type. The content of lignin in the Arabidopsis thaliana CCR1 mutant was significantly reduced, accompanied by stunted growth and reduced number of seeds [Arabidopsis thalianaC4H mutant, the growth and lignin accumulation was inhibited, apical dominance was lost and showed male sterility [CAD and CCR leading to an obvious decrease in the content of lignin in Arabidopsis thaliana and a change in lignin composition, resulting in severe suppression of plant growth and male sterility, which may be related to lack of lignin in the anther [Arabidopsis thaliana peroxidase AtPrx72 knockout mutant, which had slow growth, less branches, smaller flower and stem than the wild-type plants, as well as significantly reduced photosynthetic efficiency. However, the mechanisms of lignin affecting plant growth and development are poorly understood. Recently, Bonawitz et al. [As one of the main components of the plant cell wall, lignin confers to the function of multiple types of cells in plants tissues and organs . Lignin of seeds ,20. In Aterility . Similare anther . Herreroe anther  found thz et al.  have sugLodging resistance can prevent plant stems from bending or breaking, it is one of the most important traits that affect crop growth and grain yield . NumerouFagopyrum esculentum Moench varieties with a different lodging resistance. Peng et al.  added exCAD gene, improve the accumulation of lignin and increase the strength of the stalk, thereby enhancing the lodging resistance [+ could significantly alleviate the effect of high NH4+ on the wheat culm strength. Therefore, it was suggested that the decrease of lodging resistance induced by nitrogen fertilizer was possibly related to the inhibition of uptake of K+ which increased the lignin accumulation in the vascular bundles.In addition, nutrient elements have an important effect on plant lignin biosynthesis and lodging resistance. Silicon can enhance the expression of rice sistance . Howeversistance ,72,73. Ksistance  found thPlant cell wall is the first barrier against external hazards, one of the general reactions of plants under biotic and abiotic stresses is the accumulation of reactive oxygen species, accompanied by an increase in lignin accumulation ,75,76. TPAL, C4H and pathogenesis-related 9 (PR9) genes that associated with lignin biosynthesis were significantly up-regulated in brown plant hopper-infested insect-resistant rice varieties, suggesting that these may synergistically participate in lignin biosynthesis which regulate the insect resistance of rice [Arabidopsis thaliana root-knot nematode resistance by mediating the ethylene-dependent lignin accumulation in the roots. An insect-specific toxin peptide LqhIT2 enhanced the lignin content mediated by jasmonate and improved the leafrollers’ resistance to rice [Lignin accumulation plays an important role in the process of plant resistance to insect pests ,78. Rice of rice ,80. Wang of rice  found th of rice  found th to rice . In summArabidopsis CAD5 was highly expressed in roots with strong lignification and induced by pathogens invading Arabidopsis thaliana [DIR) was involved in the lignin deposition, overexpression of GhDIR1 significantly promoted the degree of lignification and enhanced Verticillium dahliae resistance in transgenic plants [HCT genes were identified in maize and regulated plant disease resistance by binding to NLR Rpl protein, which enhanced the expression of lignin biosynthesis pathway gene and lignin accumulation [OsAAE3 decreased lignin accumulation and increased sensitivity to rice blast and this may be related to the decrease of POD activity and expression of pathogen-related 1a (PR1) [ZmCCoAOMT2 was found to be associated with resistance to multiple pathogens, might be involved in the biosynthesis of lignin and other phenylpropanoid metabolites and regulation of programmed cell death [When plants are infected with pathogens, cell wall will accumulate a large amount of lignin, in which the content of H unit is relatively higher ,85. Incrthaliana . Cotton c plants . Two HCTmulation . Overexp1a (PR1) . In maizll death .2+, Cd2+, Pb2+, etc.) [As the first entry barrier for metal ion, cell wall is actively involved in the absorption and transport of heavy metals and the plant response to heavy metal stress ,92. Heav+, etc.) ,94 and r+, etc.) ,96.4CL, CAD, C3H and PAL genes were increased under Al stress [POD gene related to lignin biosynthesis was also up-regulated.In acidic soil, Aluminum (Al) toxicity is one of the main factors that inhibit plant growth. A typical symptom of Al toxicity is the inhibition of root growth . Tea plal stress . Other hl stress ,103,104.l stress . Bhuiyanl stress  found thThlaspi caerulescens roots were higher than that in Arabidopsis roots; tissue sections showed that the lignin accumulation in the endodermis cell wall of Thlaspi caerulescens roots was significantly higher than that of the Arabidopsis [Thlaspi caerulescens and it might be related to ion absorption and transport. Our previous study found that the deposition of lignin reduced the toxic effects of Mn, which is an important mechanism of manganese tolerance in a Mn-hyperaccumulator Phytolacca americana [2O2) was involved in the regulation of lignin monomer polymerization and lignin accumulation, this process may affect the transport of Cu from the root to the shoot in rice seedling [Lignin biosynthesis is also closely related to plant heavy metals absorption, transport and tolerance. The deposition of lignin in the cell wall of the root endoderm may inhibit the transport of heavy metal elements into the xylem or outward from the vascular bundle ,108,109.bidopsis . The resmericana . Lignifimericana . Recentlseedling .Extreme drought and high salt stress usually occur simultaneously and induce osmotic stress that causes plant cells to lose water or even die, significantly affecting plant growth and development and resulting in serious losses of crop yield ,112. LigEucalyptus urograndis and the stem apical zone of Eucalyptus globulus [CAD and COMT) and lignin content in leaves of inbred maize lines were significantly positively correlated with their drought tolerance [CCR is a key gene in lignin monomer biosynthesis pathway, under the drought stress condition, CCR1/2 were significantly up-regulated in the root elongation region of maize [Leucaena seedlings and suggested that CCR-catalyzed lignin biosynthesis may play an important role in drought stress tolerance of Leucaena. Under drought stress, the expression of CCoAOMT was obviously enhanced in the root elongation region of soybean and the accumulation of lignin in the region was also increased significantly, the increase in lignin content reduced the elongation of the root cell wall and may limit the loss of water from the roots and promote the transport of water into the elongated zone [Ipomoea batatas IbLEA14 genes enhanced transgenic callus salt and osmotic stress tolerance by increasing lignin accumulation [2O2 is one of the stresses-induced reactive oxygen species (ROS), it also plays an important regulatory role in lignin biosynthesis [Potentilla atrosanguinea superoxide dismutase (SOD) and Rheum australe ascorbate peroxidase (APX) gene enhanced the tolerance of transgenic Arabidopsis thaliana by increasing the accumulation of lignin via maintaining the level of hydrogen peroxide. MYBs have important roles in both secondary cell wall biosynthesis and abiotic stress tolerance, overexpression of Betula platyphylla BplMYB46 increased salt and osmotic stress tolerance and enhanced lignin deposition in transgenic birch, indicated that the abiotic stress response and lignin biosynthesis pathways may have a crosstalk with each other [Many studies have shown that lignin biosynthesis is enhanced under drought stress. When suffering from drought stimulation, the content of lignin was significantly increased in the stem basal zone of globulus . Under dolerance . CCR is of maize . In addiof maize  found thted zone .Recent smulation . H2O2 isynthesis ,120. Shaynthesis  found thch other . TherefoC3H gene in the cold acclimated Rhododendron leaves was notably higher than that in the control leaves, C3H may affect the cell wall rigidity and water permeability by modifying S/G ratio and participate in the Rhododendron cold resistance [EjHSF3, which was involved in fruit lignification via interacted with lignin biosynthetic regulator EjAP2-1 [Climate change affects the temperature of plant growth environment, high temperature induces the damage of biomacromolecules such as protein and nucleic acid, exacerbates membrane lipid peroxidation and disturbs the normal plant metabolism ,122. In sistance . In addi EjAP2-1 .Satsuma mandarin pericarp involved in resistance to environmental stress. Lignin content in Medicago truncatula CAD1 mutant was significantly lower than in wild type and there was no significant growth difference between the wild type and mutants at normal temperature (22 °C). However, at 30 °C, the MtCAD1 mutant growth was significantly inhibited and the growth phenotype of compensated lines were similar to the wild type. Possible reason is that reduced-lignin accumulation lead to vascular damage, thereby affecting the transpiration and causing overheating in the plant body [Gindl et al.  found thant body . p-coumaryl alcohol) [As a cheap, renewable resource, plant lignin is mostly used as an energy substance, or to develop new materials, e.g., lignin-based carbon fibers, due to the presence of phenolic hydroxyl groups and aliphatic hydroxyl groups in lignin structures. Lignin can be generally divided into three types according to the different plant species: softwood, hardwood and grass lignin. Softwood lignin consists exclusively of coniferyl alcohol, hardwood lignin consists mainly of coniferyl alcohol and sinapyl alcohol, grass lignin has three types of monomers  . In addialcohol) .The composition of the monomers has an important influence on the molecular structure of lignin, such as branching of the polymer and the degree of crosslinking with the polysaccharide . TherefoLignin is an important organic polymer which is abundant in cell walls of some specific cells. It has many biological functions such as water transport, mechanical support and resistance to various stresses. Most of the current researches on lignin in plants are focused on the regulation of lignin content through molecular biology and molecular genetics. Reducing the accumulation of lignin in energy plants can improve the production efficiency of biofuels. However, as shown above, the reduction of lignin biosynthesis can seriously affect plant growth and development, increase the risk of crop lodging, reduce plant resistance to external biotic and abiotic stresses and thus result in a serious threat to crop production.In the future, we should carefully consider the possible consequence of reducing the lignin content of the specific energy plant through the genetic modification of lignin biosynthesis genes, to maintain the normal plant physiological metabolism and improve the bioenergy efficiency, simultaneously. For some important crops, we could increase its lignin accumulation by genetic engineering, therefore enhance its resistance to external environmental factors and improve crop yields. In addition, modifying the composition and the proportion of monomers by regulating of specific lignin biosynthesis genes can provides an effective way to improve the application of lignin.


HIV-associated neurocognitive disorders occur in nearly 50% of adults with HIV. Such disorders can interfere with everyday functioning such as driving and medication adherence. Therefore, cognitive interventions are needed to address such neurocognitive disorders as well as improve everyday functioning, especially as people age with HIV.This article reports and discusses the overall rationale and development of speed of processing training, a computerized Internet cognitive training program, to improve this specific neurocognitive ability as well as everyday functioning and quality of life in adults aging with HIV. Although this protocol has been shown to improve speed of processing, everyday functioning, and quality of life in healthy, community-dwelling older adults in the advanced cognitive training in vital elderly (ACTIVE) study, its efficacy in adults aging with HIV has not been established. Nevertheless, such a cognitive intervention is particularly germane as 52%-59% of adults with HIV experience HIV-associated neurocognitive disorders (HAND), and both the frequency and severity of such disorders may increase with advancing age.The description of this longitudinal randomized controlled trial covers the following: (1) rationale for speed of processing training in this clinical population, (2) overview of overall study design, (3) eligibility criteria and HAND, (4) intervention dosage, (5) assessment battery, and (6) examination of biomarkers.The project was funded in April 2016 and enrolment is on-going. The first results are expected to be submitted for publication in 2020.Similar novel cognitive intervention approaches are suggested as they may be of value to those with HAND and may utilize similar features of this current randomized controlled trial (RCT) protocol to examine their therapeutic efficacy.ClinicalTrials.gov NCT02758093; https://clinicaltrials.gov/ct2/show/NCT02758093 (Archived by Webcite at http://www.webcitation.org/6p8C5fBCX) Combination antiretroviral therapy (cART) has resulted in improved health-related outcomes and longer life in adults with human immunodeficiency virus (HIV), with some studies estimating length of life being tantamount to those without HIV . DespiteWith such well documented neurocognitive impairments, neurocognitive aging in this group represents a major concern since by 2020, 70% of adults with HIV in the United States will be 50 years and older ,7. Thus,Speed of processing is the rate at which neurocognitive functions are performed -32. PeopFortunately, some types of computerized cognitive interventions have been shown to improve neurocognition without adverse side-effects -53. DespTargeting an intervention that specifically improves speed of processing has both theoretical and neurocognitive appeal. According to the diminished speed of processing theory ,56-59, tBased on findings from these earlier studies, this study was proposed and funded by the National Institute of Mental Health (1R01MH106366-01A1 – “An RCT of Speed of Processing Training in Middle-Aged and Older Adults with HIV”). This randomized controlled trial (RCT) study described in this article examines a well-documented intervention in the neurocognitive and gerontological literature called speed of processing training (a cognitive remediation therapy) in the population of aging adults with HIV. Specifically, there are 3 study aims: To (1) examine whether 10 versus 20 hours of speed of processing training provides differential therapeutic responses on improving this neurocognitive ability over time; (2) examine whether 10 versus 20 hours of speed of processing training will differ in therapeutic value on improving everyday functioning  over time; and (3) examine whether improvement in speed of processing and everyday functioning over time mediate improvement in quality of life indices . In doing so, these 6 key areas of the study are described: (1) rationale for speed of processing training in this clinical population, (2) overview of study design, (3) eligibilty criteria and HAND, (4) intervention dosage versus control condition, (5) assessment battery, and (6) assessment of biomarkers. Finally, the complexity of this research design is examined in relation to other cognitive interventions.Several computerized cognitive training programs have been examined in the normal geriatric population; some focus on improving functioning in a particular neurocognitive domain such as executive functioning or memory, whereas others attempt to improve more global neurocognitive functioning. Albeit, given the resources of time and effort required to engage in training programs, selecting which neurocognitive domain to be “improved” must be chosen judiciously. Whereas some experts and clinicians may focus on memory training given its salience to noticeable memory complaints, others have found targeting other domains to have more long-lasting effects that likewise produce improvements in other areas in which training was not targeted.In the advanced cognitive training in vital elderly (ACTIVE) study, researchers from 6 sites across the United States randomized normal, community-dwelling older adults  to 1 of the following treatment arms: (1) speed of processing training, (2) memory training, (3) reasoning training, and (4) no-contact control. After just 10 hours of training, those in the speed of processing training group experienced significant improvements in this neurocognitive ability. In fact, the National Institute on Nursing Research and the National Institute on Aging  announced that speed of processing training used in the ACTIVE study enabled “older people to maintain their cognitive abilities as they age,” even 10 years after training . FurtherTo promote successful aging and optimal functioning in the aging HIV population, speed of processing is a preferred target for this cognitive training intervention based on the following points. First, speed of processing is 1 of the most essential neurocognitive abilities that declines with aging, beginning in one’s 40s -71. WithA pre-post 3-group experimental longitudinal design is used . ParticiRecruitment flyers are posted at a university HIV and acquired immunodeficiency syndrome (AIDS) clinic targeting those 40+ years only. Interested potential participants call the study telephone number to be told more about the study and to determine if they meet basic eligibility requirements ; if they do, they are scheduled for a baseline assessment appointment. During this appointment, participants are consented and administered a neurocognitive, everyday functioning, and quality of life assessment. From this assessment, a HAND diagnosis is determined. Based on prior prevalence rates, it is expected over half of the participants experience HAND; only those with HAND and thus who need such a cognitive intervention are randomly assigned to 1 of the 3 treatment arms. Block stratified random assignment will minimize the risk of imbalance among randomized groups in 2 key factors: minority status and speed of processing deficiency measured by a cut off using the useful field of view test . CompletEligibility criteria specifically focus on casting a wide net to ensure that the study findings are generalizable to the larger HIV population. Albeit, given the focus on driving and neurocognition, certain inclusion, exclusion, and HAND criteria were chosen.Since driving-related factors are being examined as 1 of the outcomes of the intervention, participants must be licensed drivers when entering the study. Otherwise, participants (men and women) must be 40+ years, English-speaking, and have HAND.Due to this study being longitudinal, participants not living in stable housing  are excluded because of the challenge of scheduling follow-up visits. Potential participants are excluded if they indicate that they are planning to move away from the Birmingham metropolitan area within the next 2 years. Furthermore, participants with significant neuromedical comorbidities  are excluded; such major neurological comorbidities may confound study results and interfere with the cognitive training. These comorbidities are assessed in the telephone screen and confirmed after the baseline assessment using the university HIV and AIDS Clinic electronic medical charts. Other conditions  that could impact neurocognitive functioning or testing also necessitates exclusion; again, this information is conferred in the telephone screen and again at baseline. These typical exclusion criteria are used in many HIV neurocognitive studies ,101-104;t scores.This study focuses on HAND and speed of processing training. Typically, speed of processing declines emerge in one’s 40s  and perhBoth the speed of processing and contact-control training protocols require participants to visit the laboratory to engage in 1-hour sessions with passive supervision from a research assistant. The research assistant greets participants, helps them log onto the computer, answers questions participants may have about the training, and monitors the time participants engage in their assigned treatment arm.In the speed of processing training protocol, participants are trained to improve the speed or accuracy in which they identify and locate visual-spatial information using 5 games or exercises from the POSIT Science, which has been used in our prior studies. The games include: (1) Sweep Seeker , (2) Bird Safari , (3) Target Tracker (multiple object tracking), (4) Master Gardener (eye movements), and (5) Double Decision (UFOV). These games are automatically customized to the participants’ individual ability. The speed, difficulty, and complexity of each game is systematically increased as participants successfully master specified performance criteria. Manipulations used to increase difficulty include decreasing the duration for which the visual stimuli are presented, adding visual or auditory distracters, increasing similarity between targets or distracters, and presenting visual targets over a broader spatial expanse, which expands one’s (UFOV) and is important for driving .Studies using speed of processing training often vary from 10 to 20 hours. As mentioned in the prior study in adults with HIV, 10 hours of training was sufficient to produce significant improvement in UFOV and everyday functioning in the short-term. The NIH-funded ACTIVE study  also initially used 10 hours of speed of processing training, but additional “booster” training was found to improve the effect size and these improvements were robust over several years . A meta-In the contact-control group, participants receive 10 hours of Internet navigation training; it has been used successfully as an optimal social or computer contact-control condition for speed of processing training studies. This contact-control (sham) condition mirrors speed of processing training with the same amount of social contact with study staff and computer exposure, but does not provide any therapeutic neurocognitive benefit as we have previously observed ,81,109. Finally, a treatment fidelity checklist is used so staff can review with participants the amount of time that they have engaged in training. Furthermore, the POSIT Science software monitors the amount of time participants spend engaged in each exercise. As in the ACTIVE study, participants are considered to be trained when they successfully complete 80% of the training . This coThe project was funded in April 2016 and enrolment is on-going. The first results are expected to be submitted for publication in 2020. Study measures are assessed at baseline, immediate posttest following training, and at 2 annual follow-ups. These assessments are categorized by: (1) demographic, background, and covariate measures; (2) Aim 1, neurocognitive measures; (3) Aim 2, everyday functioning measures; (4) Aim 3, quality of life measures see . Most ofOne primary and novel focal area of this study (Aim 2) is driving and driving-related outcomes. Most adults with HIV experience some degree of neurocognitive impairment, and coupled with the lack of adequate public transportation or social support, many adults must rely on their own driving and navigational skills to carry out basic IADLs such as grocery shopping and visiting their medical providers. Thus, this cognitive intervention is expected to benefit such driving outcomes ,131-135.Driving is assessed in 3 primary ways: (1) driving simulation, (2) the driving habits questionnaire, and (3) retrospective and prospective state crash records . As mentz-scored) and summed to form a driving exposure composite score.The driving habits questionnaire  is a selz-scored) and summed to form a driving exposure composite score.Driving avoidance  is assesRetrospective and prospective vehicle state crash records are also accessed through the Alabama Department of Transportation (ADOT). As in past studies, using participants’ driver’s license numbers, a request is made to the ADOT office requesting for accident reports which are publically available records and made available for a small fee. These files contain police reports that describe the circumstances of the accident. Based upon these reports, a determination of whether participants were at fault or not is determined by 2 independent raters; when there is a lack of an agreement, a third rater makes the final determination. Thus, these state crash records can be used to determine what variables are correlated to the number of total crashes, number of at-fault crashes, and number of not-at-fault crashes. The use of actual, real life vehicle crash records provides convergent validity to the other driving measures and is also ecologically valid.Crash records can be examined both retrospectively and prospectively. Retrospectively, once the entire initial sample’s baseline data are collected, participants’ driving records can be requested for the 5 years prior to starting our study and examined to determine crash rates and neurocognitive predictors of crashes. Prospectively, once participants have completed the training in our 2-year study, it can be determined through logistic regression and hierarchical regression whether this intervention was effective in reducing the frequency and severity of vehicle crashes. Given the infrequent nature of vehicular crashes, the large sample size of this study will provide insight into whether this intervention is effective in helping adults with HAND maintain safe driving. Although obtaining information on participants’ driving exposure and driving avoidance may modify the risk of crashes for some of our participants, their combined influence can be examined over time as these variables are assessed at each time point.A unique featue of this study is the incorporation of biomarkers of brain health and brain chemistry through a separate K99 and R00 (K99AG048762) grant mechanism that uses the infrastructure of the parent R01 to examine the relationship between such biomarkers on neurocognitive and everyday functioning. In the neuroscience literature, it is clear that even basic biomarkers such as stress hormones or the amount of HIV virus in the blood can impair certain cognitive functions ; thus, mSecond, blood draws from a subset of participants (approximately 200 who are first to agree to be in this substudy) during the time of the baseline visit are also conducted to collect aliquots of blood to test for various inflammatory biomarkers  and neurotrophic factors , changes in many of which have been associated previously with HAND and HIV(+) patients with neurocognitive decline -145. AgaThis study protocol examining a cognitive remediation program is reflective of other methodologies that examine their benefits over time. As such, this study design lends itself to other types of computerized cognitive remediation programs that are online and utilize computer gaming features to improve particular neurocognitive abilities ,107. Oth


Cesarean delivery (CD) is one of the most common major surgeries performed in the United States and worldwide. Surgical techniques evaluated in well-designed randomized controlled trials (RCTs) that demonstrate maternal benefit should be incorporated into practice. The objective of this review is to provide a summary of surgical techniques of the procedure and review the evidence basis for them for the nonobstetrician. The following techniques with the strongest evidence should be commonly performed, when feasible: (1) prophylactic antibiotics with a single dose of ampicillin or first-generation cephalosporin prior to skin incision; (2) postpartum hemorrhage prevention with oxytocin infusion of 10 to 40 IU in 1 L crystalloid over 4 to 8 hours; (3) low transverse skin incision; (4) blunt or sharp subcutaneous and fascial expansion; (5) blunt, cephalad–caudad uterine incision expansion; (6) spontaneous placental removal; (7) blunt-tip needle usage during closure; (8) subcutaneous suture closure (running or interrupted) if thickness is ≥2 cm; and (9) skin closure with suture. Although the number of RCTs designed to optimize maternal and neonatal outcomes of this common procedure is encouraging, further work is needed to minimize surgical morbidity. Optimal methods for postpartum hemorrhage prevention, adhesion prevention, and venous thromboembolism prophylaxis remain ongoing areas of active research, with outcomes that could markedly improve maternal morbidity and mortality. If evidence of a surgical technique appears preferred over another, clinicians should be comfortable adopting the evidence-based technique when performing and teaching CD. Approximately 1.3 million women undergo cesarean delivery (CD) annually in the United States, making it the most common major surgical procedure performed.Not only is CD one of the most common surgeries performed worldwide, it is also one of the most unique as there are two patients to consider—the mother and the neonate. Whether performed in an urgent or nonurgent setting, atraumatic delivery of a live, vigorous neonate and subsequent minimization of maternal morbidity remains paramount and inherent in all surgical considerations. The objective of this review is to provide a summary of surgical techniques of the procedure and review the evidence basis for them for the nonobstetrician.The reasons to deliver via CD are multiple and varied depending on the circumstances of a woman's obstetric history and current pregnancy. Women with a history of previous CD, after appropriate counseling, may choose to undergo another CD in subsequent pregnancies and repeat CD accounts for a large proportion of CD indications. For example, the Consortium on Safe Labor in the United States found that a previous uterine scar was the primary indication for over half of all CDs and that 83% of women with a uterine scar are delivered by CD.Over 220 RCTs published since 1960 have been performed with regard to specific surgical techniques of CD or various generalized surgical approaches to operative technique.In addition to the aforementioned generalized CD techniques, two of the largest RCTs that evaluated optimal surgical techniques combined several steps and are worth mentioning. The CAESAR study collaborative randomized over 3,000 women to three specific technical steps: (1) single- versus double-layer uterine incision closure, (2) peritoneum closure versus nonclosure, and (3) liberal versus restricted subrectus sheath drainage.Prior to CD, the following preparation has been evaluated in RCTs: prophylactic antibiotics (7 RCTs), thromboprophylaxis (3 RCTs), preoperative vaginal preparation (2 RCTs), skin preparation (Cochrane review), and indwelling bladder catheterization (2 RCTs). There is insufficient evidence to recommend the optimal type of preoperative skin preparation. Prophylaxis with a single dose of ampicillin or first-generation cephalosporin administered prior to skin incision, however, provides the greatest reduction of maternal morbidity  with no difference in neonatal morbidity .In contrast, the RCTs that have evaluated thromboprophylaxis are largely underpowered to make specific recommendations. General hospital policies may dictate routine intermittent compression  stockings for all CDs, and universal pharmacologic prophylaxis to reduce the risk of CD-associated venous thromboembolism (VTE) remains vastly understudied. Approximately 10% of maternal deaths in the United States are associated with VTE, and the estimated CD-associated VTE rate is approximately 0.23%, twice the rate as vaginal delivery, highlighting the priority of a well-designed, appropriately powered trial in this population.Similar to mechanical stocking use, indwelling bladder catheterization remains a virtually universal practice prior to CD. However, recent data suggests that compared with noncatheterization or immediate removal, there may be a higher incidence of urinary tract infections with no significant difference in urinary retention complications in those who have indwelling bladder catheters placed.Although less routinely performed, preoperative vaginal preparation with povidone-iodine scrub is a technique that has demonstrated a reduction in postcesarean endometritis, particularly in women with ruptured membranes.The types of skin incisions available to the surgeon performing CD include midline vertical or low transverse incisions. For purposes of improved cosmesis, decreased postoperative pain, and faster overall recovery, low transverse incisions are generally preferred. Although skin incision type has not individually been compared in an RCT, the Pfannenstiel or Joel-Cohen techniques have been compared in trials of generalized CD approaches.Once the peritoneum is entered, the gravid uterus generally encompasses the entire intra-abdominal visual field. Retraction with a bladder blade and Richardson retractor may aid with visualization of the lower uterine segment. The practice of creating a bladder flap, or dissecting the visceral peritoneum of the bladder off of the lower uterine segment, has been evaluated in three RCTs with findings summarized in a recent meta-analysis.The optimal method of uterine incision and expansion has been evaluated in two RCTs and summarized in a Cochrane review. A 1- to 2-cm incision in the midlower uterine segment may used to enter the uterus. After entry, blunt, cephalad–caudad expansion has been demonstrated to decrease unintended incisional extensions and overall blood loss .20No RCTs have compared delivery techniques of the fetus. The general principles that may assist the delivery provider include: (1) ensuring an adequately sized hysterotomy incision, and (2) when the vertex is engaged in the pelvis, full flexion of the neck (chin to chest) and elevation into the hysterotomy incision. When the vertex is not engaged in the pelvis, the delivery may be accomplished with generous fundal pressure from the assistant, and in some cases delivery requires assistance with either a vacuum or forceps.Finally, in the case of breech presentation, the following technique is recommended to accomplish atraumatic delivery: (1) grasp one or both feet or, if frank breech, elevate the fetal sacrum out of the hysterotomy; (2) once elevated, rotate the fetal body to sacrum anterior; (3) apply gentle traction parallel to the maternal abdomen to the level of the fetal scapula; (4) sweep both upper extremities with abduction toward the midline; (5) use the Mauriceau-Smellie-Veit maneuver  to accomplish delivery. There are no RCTs that compare the type of uterine incision  that optimally affects delivery. In general, a low transverse uterine incision should be considered as there is no evidence to suggest difficulty of breech extraction with this type of incision and minimizes the implications a vertical uterine incision has on future pregnancies.Spontaneous (with gentle cord traction) placental removal compared with manual removal has been evaluated in 6 RCTs. Spontaneous removal is associated with a significant reduction in postoperative endometritis and may also reduce overall blood loss.The optimal postpartum hemorrhage prophylaxis remains an area of active research. Previous RCTs compared oxytocin infusion, oxytocin bolus, misoprostol, carbetocin, and tranexamic acid. These medications, either in combination or individually, have been the subject of 13 RCTs since 2013 and continue to be an active area of study. Currently, oxytocin infusion  appears to be the optimal medication to prevent postpartum hemorrhage.Hysterotomy closure using single- or double-layer suture closure remains an area of uncertainty. Although retrospective case–control studies have suggested a reduction of uterine rupture in future pregnancies with double-layer suture closure,Once the hysterotomy is determined to be hemostatic, the surgeon must then turn his or her attention to abdominal closure. If the uterus is exteriorized, it should be returned to its anatomic position. Reassessment of hemostasis once the uterus is replaced is prudent. Intra-abdominal irrigation with warm normal saline to remove blood clots and debris does not appear to reduce morbidity from infection but may increase maternal intraoperative nausea, as evaluated in one RCT.Peritoneal closure compared with nonclosure remains an active topic of research and has resulted in 19 RCTs, 2 meta-analyses, and a systematic review. Evidence supporting closure versus nonclosure depends on the outcome measure studied. However, there is no long-term outcome data on the most clinically relevant outcome measure—adhesion formation. As such, closure versus nonclosure of the peritoneum remains the preference of the surgeon at this time.Fascial closure is accomplished using absorbable suture in a running fashion. The optimal suture material has not been compared in an RCT. However, blunt-tip needle compared with sharp needle for closure of all tissue layers during CD was evaluated in one RCT and also was included in an analysis in a Cochrane review that also included other types of surgery. Notably, blunt-tip needle use significantly reduces the overall risk of glove perforations and percutaneous exposure incidents and should be routinely available and used in all CDs.Subcutaneous skin closure (running or interrupted technique) is recommended if the tissue thickness exceeds 2 cm based on the available evidence of 11 RCTs that compared subcutaneous skin closure versus nonclosure, with or without drain placement. Finally, based on a meta-analysis of 3,112 women in 12 RCTs, skin closure with absorbable suture rather than metal staples is strongly recommended as this method significantly decreases wound morbidity (particularly wound separation) without a difference noted in pain, patient satisfaction, or cosmetic results.CD remains the most common major abdominal surgery performed in the United States. As such, the surgical techniques evaluated in well-designed RCTs that demonstrate maternal benefit should be incorporated into practice.


Cytotoxic immunity relies on specialized effector T cells, the cytotoxic T cells, which are endowed with specialized cytolytic machinery that permits them to induce death of their targets. Upon recognition of a target cell, cytotoxic T cells form a lytic immune synapse and by docking the microtubule-organizing center at the synaptic membrane get prepared to deliver a lethal hit of enzymes contained in lytic granules. New insights suggest that the directionality of lytic granule trafficking along the microtubules represents a fine means to tune the functional outcome of the encounter between a T cell and its target. Thus, mechanisms regulating the directionality of granule transport may have a major impact in settings characterized by evasion from the cytotoxic response, such as chronic infection and cancer. Here, we review our current knowledge on the signaling pathways implicated in the polarized trafficking at the immune synapse of cytotoxic T cells, complementing it with information on the regulation of this process in natural killer cells. Furthermore, we highlight some of the parameters which we consider critical in studying the polarized trafficking of lytic granules, including the use of freshly isolated cytotoxic T cells, and discuss some of the major open questions. Cytotoxic T lymphocytes (CTLs) armed with lytic machinery exert a non-stop patrol of our body to identify and eliminate target cells with potentially “dangerous” phenotype. CTLs bear T cell receptors (TCRs) through which they bind antigenic peptides presented on the major histocompatibility complex (MHC) molecules of target cells. In this way, they successfully discriminate between healthy cells and those presenting non-self peptides, typically of neoplastic or microbial origin. As consequence of non-self recognition, CTLs attack and lyse malignant and infected cells. In this context, an impaired functioning of CTLs may lead to immune evasion of tumors and the insurgence of chronic infections. Hence, defining the mechanisms underlying CTL-mediated killing could provide essential insights into our understanding of immune pathology.The immune synapse of CTLs represents a highly organized system of intercellular communication. Its assembly is initiated upon CTL recognition of a cognate target, toward which the CTL rapidly polarizes Figure A. WithinThe polarization and exocytosis of lytic granules at the immune synapse are driven by local signaling, which triggers two major polarization events, namely (i) the polarization and docking of the MTOC at the synapse and (ii) the polarization of lytic granules toward the docked MTOC, followed by granule exocytosis or “degranulation,” which results in the focal release of granule contents. Notably, at variance with the mechanisms regulating MTOC translocation, the signaling events that regulate lytic granule polarization remain as yet to be fully characterized. Understanding how granule convergence at the synapse is regulated could shed substantial insight into the mechanisms driving CTL dysfunction.Here, we present an overview of the mechanisms that orchestrate the polarized transport of lytic granules in CTLs and incorporate some relevant information on the signaling of natural killer (NK) cells for a comparative analysis of polarizing signals in these two types of cytotoxic lymphocytes. We also discuss recent experimental evidence describing dysfunctional non-polarized immune synapses. Finally, we comment on the parameters of experimental setups that we consider important for the study of polarized trafficking in CTLs and highlight the major open questions concerning its orchestration.Many of the signaling pathways contributing to the assembly of the CTL synapse have been dissected Table . Loss-ofSignaling initiated upon target recognition by the CTL is dictated by the TCR, which recognizes antigenic peptides presented by MHC class I molecules on the target and by costimulatory signals triggered through the engagement of CTL surface receptors by various ligands expressed on the target. Signaling studies on planar lipid bilayers coated with defined molecular ligands allowed to establish that essentially only two types of molecules may be triggered on a CTL to induce synapse formation . These a+ T cell synapse formation is that the TCR and some of the costimulation/inhibitory molecules are spatially segregated during synapse assembly. This stands true also for the mature immune synapse of CTLs. Upon the initiation of CTL synapse assembly, the TCRs and proximal signaling molecules of the TCR pathway concentrate into the central area of the synapse [known as the central supramolecular activation cluster (cSMAC)], while LFA-1 and other adhesion molecules segregate into a ring which surrounds the cSMAC [peripheral supramolecular activation cluster (pSMAC)] (An important notion that has emerged from the first molecular studies of CD4(pSMAC)] –12. The (pSMAC)] . The thr(pSMAC)] , the pSM(pSMAC)] , while t(pSMAC)] .Interestingly, the formation of a mature immune synapse with signaling molecules arranged in the SMAC pattern does not constitute a pre-requisite for successful killing by CTL. It has been shown that as few as two to three peptide/MHC complexes on the surface of the target can trigger cytotoxicity , 16. In In this review, we will summarize the current knowledge on the TCR and costimulatory signaling pathways in CTLs and discuss their contribution to the regulation of polarized synaptic secretion Figure B. For thProximal TCR signaling at the cSMAC is initiated by the two Src family kinases Lck and Fyn . Lck act2+ signaling, and cytoskeletal reorganization , which recruits numerous signaling effectors to form a multiprotein complex termed the LAT signalosome . The LATnization .2+ influx (2) leading to the formation of the lipid second messengers diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). The local DAG gradient promotes the synaptic recruitment of protein kinase C (PKC) isoforms, namely novel PKCs including PKCθ, PKCε, and PKCη, which participate in the proximal signaling inducing MTOC polarization  membrane and activates Ca2+ release from the ER into the cytoplasm , the most potent Ca2+-releasing second messenger known, is also produced following TCR stimulation and crucially contributes to the signaling pathway operating during CTL degranulation  , CD2 al, and CD12, which promote the recruitment of LFA-1 to the immune synapse and help it to acquire a stable high-affinity conformation [reviewed in Ref.  from PIP2, which has been identified as a key event promoting the spatial segregation of F-actin to the dSMAC ring . Thus, WASP could be recruited to the synapse by two mechanisms: through LAT-SLP76 signaling  and throThe SLAM family members have been extensively studied in CTL functioning due to their involvement in the X-linked lymphoproliferative (XLP) syndrome , 94. XLPAlthough SLAM receptors are important for CTL-mediated cytotoxicity , 104, thInterestingly, studies on human and murine NK cells have demonstrated that LAT is constitutively associated with 2B4 in glycolipid-enriched membrane microdomains and has a critical role in SLAM-mediated cytotoxicity –110. Conin vitro after their isolation from human or mouse donors. CTL expansion in this case is usually required due to a relatively low percentage of effector cytolytic cells among the total pool of CD8+ T cells [in healthy human donors 10–15% . It has become evident that culturing CTLs in IL-2-supplemented medium may alter signaling requirements for CTL killing when compared to freshly isolated effector CTLs. In particular, this knowledge emerged from studies on CTLs from patients and mice with genetic mutations that affect CTL functioning. For instance, function-disrupting mutations in Munc18-4, which regulates lytic granule fusion with the plasma membrane, abolish cytotoxicity in freshly isolated CTLs due to a loss of their ability to degranulate  O+ cells . Effecto11 and PLCβ, instead of Lck and PLCγ1, at least as shown for CD4+ T cells  that are able to cross-link the MHC class II molecules on target cells and the TCRs on CTLs  . In this T cells , 125. Neely used , 35, 52.The second system of polyclonal stimulation commonly employed to work with human CTLs involves the use of Fc receptor-bearing target cells  loaded with CD3-specific monoclonal antibody. Antibody-coated targets are recognized by the CTLs through direct binding of the TCR to the antibody, thus promoting cytotoxic killing in the so-called “redirected” assay . Of noteAlthough both systems appear to work well with freshly isolated CTLs, the use of MHC class I-peptide system is obviously more physiologically relevant as it may allow to assess the functioning of CTLs with natural ligands of different affinity and specificity. The solution to overcome the limitation of rare individual clones in the total CTL population came with the development of systems to clone and express recombinant TCR receptors, derived from naturally occurring CTL clones, in polyclonal freshly isolated CTLs . For insDirectionality of lytic granule transport along microtubules represent fine means to regulate immune synapse assembly in CTLs and NK cells, thus representing a basic factor in immune regulation. However, the mechanisms defining granule ability to travel bidirectionally along microtubules and, most importantly, the signaling pathways regulating this process are largely elusive. To address this question of outstanding scientific importance, we believe that future research on CTLs should focus on some specific issues. First, it would be of high relevance to identify surface receptors that trigger the signaling pathways driving granule translocation toward the MTOC. So far, it emerges that TCR triggering alone could not be sufficient to induce granule polarization, at least in freshly isolated human CTLs , suggestin vitro expanded CTL blasts (Second, lysosomes have emerged as important signaling hubs implicated in the regulation of multiple cellular processes including metabolic signaling and autophagy . Along tL blasts , 137. WeFinally, it is becoming widely accepted that stimulation with cytokines is capable of reorganizing the molecular and signaling network of granule trafficking and secretion in cytotoxic lymphocytes . HoweverAK and VZ wrote the initial draft for the manuscript which was then revised and contributed by CB.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 reviewer AA declared a past collaboration with one of the authors CB to the handling editor.


Improving the ability of plants and plant-associated organisms to fix and assimilate atmospheric nitrogen has inspired plant biotechnologists for decades, not only to alleviate negative effects on nature from increased use and availability of reactive nitrogen, but also because of apparent economic benefits and opportunities. The combination of recent advances in synthetic biology and increased knowledge about the biochemistry and biosynthesis of the nitrogenase enzyme has made the seemingly remote and for long unreachable dream more possible. In this review, we will discuss strategies how this could be accomplished using biotechnology, with a special focus on recent progress on engineering plants to express its own nitrogenase. Strategies to improve the ability of plants to fix atmospheric dinitrogen are discussed, with a special focus on recent advances in biochemistry, synthetic biology and plant biotechnology. The reason for this is that most organisms, including all eukaryotes, can only assimilate reactive nitrogen (Nr), such as oxidized  or reduced  nitrogen species. Only a small groups of prokaryotes (bacteria and archaea), collectively referred to as diazotrophs, can convert N2 into biologically available nitrogen (NH3) in a process called ‘nitrogen fixation’.The human body consists of 3% nitrogen, an essential constituent of biological molecules such as amino acids (proteins), nucleic acids (DNA and RNA), adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide. Although 78% of the atmosphere is composed of dinitrogen gas  is produced from H2 and N2 that react over a catalyst at high temperature and pressure. Developed by Fritz Haber and taken to industrial/commercial scale by Carl Bosch during 1908–1913, the Haber–Bosch process enabled the Green Revolution about 40 years later, and has been estimated to have permitted the human population to exceed 3 billion people  is performed by a group of prokaryotes belonging to the bacteria or archaea domains. No eukaryote is capable of converting N2 into a biologically active N species. Depending on the habitat in which these diazotrophic organisms grow, they can be divided into three groups: (i) free living (e.g. Azotobacter vinelandii), (ii) symbiotic, mainly bacteria living within plant root nodules, such as Rhizobium in legumes, and in animals such as the digestive tracts of termites and coral reef sponges  and Component 2 (dinitrogenase reductase) , others are facultative anaerobes (Klebsiella oxytoca), meaning that they can grow both aerobically and anaerobically, but only fix N2 under anaerobic conditions  which ensures that O2 levels are sufficient for respiration  and has an active site metallocluster called FeMo-cofactor (FeMo-co) (Shah and Brill nifH gene product.Three types of dinitrogenase (Component 1) exist in nature, classified according to the active site cofactor capping metal: the most abundant and ecologically relevant molybdenum (Mo) nitrogenase, and the alternative vanadium (V) and iron-only (Fe) nitrogenases  proposed to differ in having V or Fe at the position of Mo in FeMo-co  to the non-diazotroph Escherichia coli further encouraged such thinking  from Pseudomonas stutzeri A1501 to the aerobic root-associated beneficial bacterium P. protegens Pf-5   when forming symbiotic arbuscular mycorrhiza  8 electrons and the hydrolysis of 16 ATP  and harbor an [Fe-S] cluster assembly machinery similar to the bacterial Isc system  as model organism for initial testing of the functionality of eukaryotic Nif proteins. Yeast is a fast-growing organism that is easy to transform, and it can be grown under various conditions with different O2 concentrations, making it an excellent host when testing Nif protein functionality and O2 susceptibility.Alterations in for example gene structures, codon-usage and promoters are different between (and within) prokaryotic and eukaryotic cells. Synthetic biology is therefore required for fast and high-throughput transfer of genes from a diazotrophic organism into a plant cell. In this sense, recent development of (affordable) gene synthesis has been crucial. However, as transformation of plants and plant cells is time-consuming, and as mitochondria Fe-S cluster biosynthetic machineries are similar in plants and yeast, we are using Finally, it is important to stress that this work-flow constantly adapts to new knowledge. As nitrogenase synthesis and activity requires the action of a multitude of gene products, new information has direct implications on the other components of the system.2 fixation machinery to plants requires efforts and inputs from several disciplinary fields, highlighting the need for collaborations. In our experience, each one faces its own problems that will be outlined in the following sections  and nifUSVWZM . Second, the refactored nif gene cluster only generated about 7% of the wild-type (WT) activity, highlighting difficulties when artificially reproducing biological systems that are heavily regulated in their original hosts.The expression level of et al.nifEN) resulted in strikingly low activity. Similar studies from other researchers confirmed the advantages of synthetic biology to engineer nitrogenase in heterologous hosts , resulting in high expression levels  activity and when the plants had previously been incubated at subambient O2 levels  location of the engineered nitrogenase is probably the most important issue to address regarding plant cell biology. Spatial separation of photosynthesis and Ns Maliga . In this2 fixation, using promoters whose activity follows the circadian rhythm. This has been tested in cyanobacteria, where the Cyanothece N2 fixation genes are upregulated during the dark period  were studied to find transcription factors that could separate expression of nif genes from photosynthesis when transferred from Cyanothece to Synechocystis  will be sufficient for nitrogenase maturation is not known. Cellular concentrations of the structural proteins NifH and NifDK under N2-fixing conditions in A. vinelandii is about 100 and 50 μM, respectively  or N2 fixation (dark) has been alleviated by a process called Fe conservation, where metalloenzymes are daily synthesized and degraded  NifDK nitrogenase component, 96 yeast strains with mitochondria targeting of NifH, NifDK, NifU, NifS, NifM, NifB and NifEN were generated  was also targeted to yeast mitochondria  are important tools. Until now, mitochondria appear to be the organelle of choice, as aerobic yeast cultures could accumulate active NifH (Lopez-Torrejon


Coffea arabica L. and the native tree community of secondary forests regrowing after the abandonment of coffee plantations is important because, as a non-native species in the Neotropics, coffee can outcompete native species, reducing diversity and forests ecosystem services. We aimed to answer three questions: 1) Does coffee regeneration in secondary forests differ between shaded and unshaded abandoned plantations?; 2) How is coffee basal area related to structural attributes, species diversity and composition of the native community?; and 3) Do the relationships between coffee and native community differ between tree and sapling components? We sampled the tree and sapling components in a seasonal tropical dry forest that were previously used as shaded and unshaded coffee plantations. Coffee was the most important species in the sapling component of shaded systems, but was almost absent in unshaded ones. Coffee basal area was negatively related with the native density and absolute species richness of the sapling component; and was negatively related with tree density, and positively related with the percentage of pioneer individuals of the native tree component. Our results indicate that coffee persists in secondary forest communities even after more than 70 years of shaded-coffee plantations were abandoned, potentially reducing density and diversity of native species. Despite limitations, which hinder more general conclusions on coffee invasiveness in Brazilian secondary tropical forests, our results indicate that coffee is a strong competitor in the studied secondary forests and provide important insights for future research on this topic.Understanding the relationships between  Secondary forests account for most forest cover in the world , and theCoffea arabica L. is widespread in tropical countries [The association of secondary forests and ountries , 13. In ountries . In the ountries –17. Manyountries . AlthougA central but yet understudied question is whether coffee can affect Neotropical secondary forest communities. Coffee agroforestry systems provide important ecosystem services such as carbon storage , and habIn addition to changes in forest structure and species richness, it is expected that coffee would affect forest composition  . For insCoffea arabica populations and the native communities (tree and sapling components) of secondary forests regrowing after the abandonment (> 70 years) of shaded and unshaded coffee plantations. We addressed three main questions: 1) Does coffee persistence in secondary forests differ between shaded and unshaded abandoned plantations? We hypothesized that after abandonment, the relative importance of coffee  would be higher in forests growing in previously shaded areas because their environmental conditions would be more similar to forests where coffee is native since the plantation abandonment, while unshaded coffee will experience the hash environmental of initial stages of forest succession before get fully shaded; 2) How is coffee basal area related to the structure, species diversity and composition of the native community? We hypothesized that coffee (a non-native species) would be negatively related with native forest attributes ; 3) Do the relationships between coffee plants and the native community differ between tree and sapling components? We hypothesized that the relationship of coffee (an understory species) and the native community is size-dependent, being stronger for the sapling component than the tree component.The aim of this study was to evaluate the relationship between This study was conducted in three patches of seasonal dry forest in Juiz de Fora municipality, Minas Gerais state, Brazil. Two of them are located in the Botanical Garden of the Federal University of Juiz de Fora  and the other in the Mariano Procópio Park . Sampling permission in BGJF was granted by Federal University of Juiz de Fora and permission in MPP was granted by the administration of MPP. The distance between BGJF and MPP is 2.3 km and both areas are conservation units surrounded by an urban matrix. During the sampling, no signs of cattle grazing, fire and/or selective logging were observed. These forests experience a homogeneous climate, defined as warm temperate climate (Cwb Megathermic climate of Köppen), with warm and rainy summers and dry winters. Mean annual rainfall is 1516 mm and mean annual temperature is 19°C . AltitudPiptadenia gonoacantha (Fabaceae) due to the ability of this species to fix nitrogen, its longevity, and provide a broad canopy. The preparation of a shaded coffee plantation included the elimination of all the native vegetation with exception of relatively few canopy individuals. In the 1930s, Brazil experienced an economic crisis in coffee cultivation, and most of coffee plantations located in the region of Juiz de Fora were abandoned [In the past (> 70 years ago), coffee cultivation was an important economic activity in the region of Juiz de Fora  and the bandoned , allowinbandoned –39. AlthTo evaluate the tree component, we allocated 25 randomized sample plots (20 x 20 m) in BGJF-1 (shaded coffee) and BGJF-2 (unshaded coffee), totalling 1-ha sample in each area. In MPP (shaded coffee) we allocated 10 randomized sample plots (20 x 20 m) totalling 0.4 ha sample. We sampled 10 plots instead of 25 plots in MPP because it is a small forest fragment (5 ha).We randomized sample plots in the core areas of each forest, so as to avoid edge effect in our plots. In each plot, the tree component included all living trees with stem diameter at breast height  ≥ 5 cm. Trees were tagged, identified to species level and their diameter was measured. To evaluate the sapling component, sub-plots of 5 x 5 m were allocated inside each tree component plot (southeast corner), and all saplings with DBH ≤ 5 cm and height ≥ 1 m were tagged, identified to species level and their diameter was measured.Evaluating species into ecological groups is useful to understand how communities differ in species composition  . In foreerror), reducing or even removing the spatial correlation among plots [We evaluated the parameters of species relative importance values  in each forest patch and each community component. To test whether coffee regeneration RIV parameters differ between coffee cultivation systems (shaded vs unshaded) we used generalized linear mixed models (GLMM), including patch size as a random effect (to account for the possible confounding effect of patch size in our results). To account for the nestedness of plots within forest patches, we performed simultaneous autoregressive (SAR) models, assuming spatial autocorrelation among plots and including a second error term in the GLMM. SAR models evaluate the spatial dependence in the residuals of GLMM , absolute species richness (S), rarefied species richness (S’), species diversity  and percentage of pioneer individuals . We used rarefied species richness to account for the confounding effect of tree density in species richness [error models. We used coffee basal area as a predictor because basal area better reflects species relative biomass, which is a better indicator of plant performance than abundance (i.e. the effect of one larger coffee tree is stronger than many coffee saplings). We did not include unshaded patch in the bivariate relationships between coffee and native forest attributes because coffee was absent in most plots , basal area  and with the following packages: lme4 , vegan , spdep , and ggpA total of 4,250 individuals (189 species and 48 families) were sampled in the tree component and 2,400 individuals (148 species and 40 families) in the sapling component . We founWe evaluated how coffee populations are related to structural and diversity attributes of secondary forests growing after abandonment of shaded and unshaded coffee plantations. We found that coffee population persisted (after more than 70 years of abandonment) with high basal area, density, and frequency in the understory of shaded plantations but was almost absent in the unshaded plantation. We found that coffee was negatively related with native density (tree and sapling components), and absolute species richness (sapling component), and was positively related with the percentage of pioneer individuals (tree component).e.g. wind intensity, desiccation, temperature variation and soil degradation), and by increasing nutrient recycling through litter decomposition [We hypothesized that the relative importance of coffee would be higher in secondary forest growing in abandoned shaded plantations because coffee trees are under similar conditions to its native habitat since plantation abandonment. Conversely, although a shaded environment will be achieved in unshaded plantations areas after decades of abandonment, coffee trees experience a harsh environment during initial stages of forest succession before they get fully shaded. We indeed found higher coffee relative basal area, density and frequency in shaded plantations than unshaded plantations Figs  and 2. Sposition –59. Finaposition , 61, attCoffea arabica.We acknowledge that differences in coffee performance under shaded and unshaded conditions could be related to differences in coffee varieties or cultivars, but unfortunately we do not have this accurate information. Yet, considering the interviews with local residents and landowners, the long-time interval since plantations were abandoned (>70 years), and that research on high-yielding and disease-resistant coffee varieties only occurred from the late 1960s, we assumed that coffee plants in all studied forest patches belong to the same cultivar of Our results indicate that coffee is a strong competitor in secondary tropical forests growing after the abandonment of shaded plantations, and highlight the importance of understanding abandoned plantation legacies, because the species cultivated in the original system can persist for a long time and become “ghosts of cultivation past” .We hypothesized that coffee basal area (a non-native species in the Neotropics) would be negatively related with native forest attributes, and that these relationships would be size-dependent, being stronger for the sapling component (SC) than the tree component (TC). We indeed found significant relationships between coffee basal area and forests attributes but we found little evidence to support our size-dependent hypothesis, because coffee was significantly related with two out of six attributes for both tree component (tree density and percentage of pioneer species) and sapling component (density and absolute species richness). Our results contrast with many studies that found null or even positive effects of coffee plants on native tree communities , 27, 53,Euterpe edulis, Pseudopiptadenia contorta and Siparuna guianensis, that mostly occurs where coffee basal area is low, see Piptadenia gonoacantha . Our findings that coffee is still present in high abundance  in secondary forests from shaded plantations areas after more than 70 years, highlight coffee legacy and persistence in those forests. We also found lower density and diversity of saplings and higher number of pioneer trees where coffee is dominant, suggesting that coffee is a strong competitor in secondary forests, potentially overyielding native species and taking advantage of degraded forests. As a consequence, some important ecological process of secondary forests can be altered, such as species composition, light requirements for regeneration, forest dynamics and aboveground biomass stocks and carbon residence. Moreover, they suggest the need for management of coffee in abandoned shaded coffee plantations because coffee may be negatively related to forest regeneration. Studies on a large-scale will be important to understand the role of this species in such forests and to manage these areas for conservation and sustainable use of agroforestry systems.We acknowledge that the observed relationships do not necessarily mean causation, and that the small number of replicates and the experimental design does not allow for strong conclusions about the invasive potential of S1 Table(XLSX)Click here for additional data file.S2 TableCoffee density and basal area were calculated for both TC and SC.(DOCX)Click here for additional data file.