Diverse and flexible cortical functions rely on the ability of neural circuits to perform multiple types of neuronal computations. GABAergic inhibitory interneurons significantly contribute to this task by regulating the balance of activity, synaptic integration, spiking, synchrony, and oscillation in a neural ensemble. GABAergic interneurons display a high degree of cellular diversity in morphology, physiology, connectivity, and gene expression. A considerable number of subtypes of GABAergic interneurons diversify modes of cortical inhibition, enabling various types of information processing in the cortex. Thus, comprehensively understanding fate specification, circuit assembly, and physiological function of GABAergic interneurons is a key to elucidate the principles of cortical wiring and function. Recent advances in genetically encoded molecular tools have made a breakthrough to systematically study cortical circuitry at the molecular, cellular, circuit, and whole animal levels. However, the biggest obstacle to fully applying the power of these to analysis of GABAergic circuits was that there were no efficient and reliable methods to express them in subtypes of GABAergic interneurons. Here, I first summarize cortical interneuron diversity and current understanding of mechanisms, by which distinct classes of GABAergic interneurons are generated. I then review recent development in genetically encoded molecular tools for neural circuit research, and genetic targeting of GABAergic interneuron subtypes, particularly focusing on our recent effort to develop and characterize Cre/CreER knockin lines. Finally, I highlight recent success in genetic targeting of chandelier cells, the most unique and distinct GABAergic interneuron subtype, and discuss what kind of questions need to be addressed to understand development and function of cortical inhibitory circuits. Figure 1). Therefore, unraveling “subtype-specific” development, connectivity, and function of GABAergic interneurons will provide clues toward understanding how functional cortical circuits are wired and how the brain integrates information and generates outputs, which have been central issues in neuroscience.The mammalian neocortex is a highly evolved organ that plays a critical role in higher order brain functions such as perception, learning, memory, and behavioral outputs. It executes many kinds of neuronal computations through complex and delicate interactions between distinct types of neurons in the cortical and subcortical regions. Among many neuronal signals, excitatory and inhibitory neurotransmissions are the most fundamental components that shape activity patterns of neural networks. In particular, cortical inhibition locally provided by GABAergic interneurons has a key role in not merely overall balance of network excitability but also synaptic integration, spike timing, and synchrony of a neuronal ensemble . The varTable 1). In parallel, several methods to introduce these genetic tools into intact brains, including viral delivery, mouse genetics, and electroporation have been developed. Analysis methods such as new microscopy  and high-throughput sequencing have also evolved, further enhancing the value of genetic probes and effectors. However, especially in the case of studies in GABAergic interneurons, these tools need to be expressed in specific subtypes for clear interpretations. Until recently, such targeted expression was not possible because there has been no systematic strategy that enables reliable expression of exogenous genes in GABAergic interneuron subtypes. This technical limitation has hampered a comprehensive understanding of development and function of GABAergic local circuits.In the past decade, an explosive development of genetic tools to investigate neuronal circuits molecularly, anatomically, and physiologically has been made. These tools allow us to obtain molecular expression profiles, identify connected neuronal populations, visualize signaling events and voltage changes, and optically manipulate neuronal activity , the most distinct GABAergic interneuron subtype.Figure 1). Although researchers have tried to find a simple relationship between cell types distinguished by distinct criteria, one cell type defined by expression of a certain gene can exhibit several types of physiological properties, and vice versa. However, ultimately classification of GABAergic interneuron subtypes based on the expression of a combination of genes may reveal distinct cell types, since physiology and connectivity are likely explained by sets of genetic programs. Although it is still far from the stage that truly pure subtypes are described by gene sets, there are several markers that can delineate broad subclasses of GABAergic interneurons. Here I introduce major subtypes of GABAergic interneurons, which are classified by gene expression of calcium-binding proteins and neuropeptides, and representative minute cell types within each subtype.To understand development, organization, and operation of GABAergic circuits, it is critical to take cellular diversity into account. Many lines of evidence have suggested that there are a myriad of GABAergic interneuron subtypes classified by physiology, morphology, connectivity, and gene expression . Most of PV-expressing interneurons are so called basket cells, which can be further subdivided by size of the cell body , and dendritic and axonal projection . Feedforward inhibition mediated through FS PV-expressing basket neurons can be found in several cortical networks including thalamocortical, translaminar, and interareal circuits . It is widely accepted that SOM neurons are classified as dendrite-targeting cells with respect to the output connectivity . Representative cell types that belong to SOM-expressing neurons include Martinotti cells in the neocortex, which project ascending axons that horizontally bifurcate in layer 1  is a neuropeptide, which is expressed by 30% of cortical interneurons. SOM-expressing interneurons have little overlap with PV-expressing interneurons , the calcium-binding protein. SOMectivity . In L2/3 neurons . Another neurons . X94 is  neurons .Figure 2A). 5HT3aR neurons are very heterogeneous physiologically, anatomically, and biochemically but roughly divided into two subclasses, vasoactive intestinal peptide (VIP) neurons (see below) and non-VIP neurons . Non-VIP 5HT3aR neurons corresponding to 60% of the 5HT3aR neurons, include Reelin-expressing neurons. The Reelin-positive neurons are a primary population (more than 80%) of non-VIP 5HT3aR cells . A fraction of the Reelin+/VIP-/5HT3aR+ neurons contains neurogliaform cells (NGFCs), which have small and round somata with a dense axonal plexus containing small presynaptic boutons . NGFCs can cause slow and sustained inhibitory postsynaptic potentials (IPSPs) in target neurons within the area covered by their axonal plexus, through the concomitant activation of slow GABAA and GABAB receptors (The 5HT3a receptor (5HT3aR) is the inotropic serotonergic receptor, which is expressed in most, if not all, GABAergic interneurons that express neither PV nor SOM and comprise about 30% of total cortical interneurons . A significant number of VIP neurons coexpress CR and display bitufted/bipolar morphology . By physiological characterizations, these neurons are usually referred to as irregular-spiking cells . These results may suggest that VIP neurons disinhibit pyramidal neurons. On the other hand, as SOM neurons inhibit PV neurons as one of their postsynaptic targets . Just like PV neurons, CCK basket cells are thought to control the phasing and synchronization of neural ensembles . Notably, type I neurons selectively spike during slow wave sleep when most cortical neurons are relatively silent (Nitric oxide synthesized by nitric oxide synthase (NOS) is a retrograde signaling molecule that modulates neuronal transmission, although its roles in GABAergic interneurons are largely unknown. nNOS is broadly expressed in the cerebral cortex during development  but is cy silent . These fy silent .+ (cut-like homeobox 2 protein) RGCs produce later-born upper-layer neurons, while earlier-born lower-layer neurons are generated from Cux2- RGCs . The other mechanism that explains production of different neuronal types is temporal restriction of competence of a common progenitor to differentiate into certain cell types . Taken together, strategies to diversify neuronal cell types include formation of distinct progenitor domains, lineage-dependent cell fate determination, progressive changes in cellular competence to give rise to certain cell types, and intrinsic/extrinsic controls in postmitotic neurons to accomplish specific terminal differentiation. At present it still remains unclear to what extent GABAergic interneurons utilize these strategies to generate a variety of cell types, but it is likely that all the possibilities are used in combination. Below I will outline our current understanding in origins of GABAergic interneurons and molecular determinants for subtype specification.In the nervous system, the diversification of neuronal cell types is a common strategy to ensure functional complexity and flexibility of the neuronal networks. Cortical GABAergic interneurons display the highest degree of heterogeneity, which brings up a lot of important and exciting questions but has hampered fine experiments needed to gain definitive conclusions. Understanding cellular and molecular mechanisms that generate diverse subtypes of GABAergic interneurons is critical, not only for unraveling mechanisms for the subsequent assembly of neural circuits, but also for developing genetic methods to visualize and manipulate distinct subtypes at any stage from birth to functional maturation. Although our knowledge on mechanisms for subtype specification of GABAergic interneurons is still very limited, there is highly suggestive information regarding cell fate determination mainly coming from other systems such as motor-related neurons in vertebrates, neurogenesis of cortical excitatory neurons, and neurogenesis in fruit flies. In particular, because of their relatively simple cellular organization, as well as a rich repertoire of molecular markers that specifically label neuronal subtypes, the motor-related neurons in the spinal cord have provided ideal experimental systems to study cell type specification . What onx2- RGCs . Cux2+ Rprofiles . These rll types . This coFigure 2A).Most, if not all, cortical GABAergic interneurons are generated from the ventral telencephalon including the medial ganglionic eminence (MGE), the preoptic area (POA), and the caudal ganglionic eminence (CGE), in non-primate mammals. Immature GABAergic interneurons tangentially migrate toward the cortex . How different classes of interneurons are produced within the MGE is largely unknown but recent studies have demonstrated that each subtype of interneuron has preferential spatial and temporal origins. The systematic in situ hybridization analysis for many TFs expressed in the MGE identified five distinct progenitor domains called pMGE1 to pMGE5. Each of these domains expresses a unique combination of genes, and led to the hypothesis that each progenitor domain generates different subtypes of GABAergic interneurons . Moreover, it has been shown that the sonic hedgehog (Shh) signaling effectors such as Gli1, Gli2, Ptch1, and Nkx6.2 are enriched in the dorsal MGE . More recent results based on the clonal analysis have demonstrated that clonal neurons are born isochronically and typically form a cluster within one or two neighboring layers rather than in a columnar manner, favoring the presence of lineage-restricted progenitors . Genetic fate mapping experiments using Nkx5.1-Cre mice revealed that roughly one third of the progenies derived from these progenitors express neuropeptide Y (NPY) and none of them express representative interneuron markers such as PV, CR, SOM, VIP, and nNOS . A genetic fate mapping study with Dbx1-Cre mice indicated that Dbx1-expressing progenitors in the POA2 produce major subtypes of cortical GABAergic interneurons including PV, SOM, Reelin, CR, NOS, and VIP neurons, which share neurochemical properties with those derived from the MGE or the CGE . As mentioned in the previous section, 5HT3aR-positive neurons express different combinations of CR, VIP, NPY, and Reelin  and the MGE with ambiguity. However, recent studies have demonstrated that the CGE can be molecularly delineated with a unique set of genes , and divided into several progenitor domains . What wad Reelin .Figure 2C), which is induced and maintained by the activity of Shh  and the subpallium . Upon patterning of the neuroepithelium along the dorsal–ventral axis through the actions of morphogens such as Shh  and boney of Shh , 2005.A loss-of-function study revealed that in Nkx2.1 null mutant mice, the MGE is mis-specified to the fate of the LGE and as a consequence, more than half of the cortical interneurons including SOM-, NPY-, and calbindin-expressing neurons are lost . Since NFigure 2C). In the mature cortex, the expression of Lhx6 is confined to PV- and SOM-expressing neurons, which are produced exclusively in the MGE  of the MGE . GeneticFigure 2C). Cortical interneurons in Dlx1/2 and Dlx5/6 double mutants show common defects in tangential migration  are widely expressed in subpallial progenitors of the LGE, MGE, and CGE , and conigration . Howeverigration . In the igration . Consistigration .Transcriptional mechanisms that control the fate determination of the CGE have remained largely elusive. Gsh2 is a homeodomain TF that is normally expressed in the dorsal LGE and the CGE . ConditiTable 1). For more details regarding these techniques, other reviews should be referred.Recent advances in molecular tools as well as genetic strategies to interrogate structure and function of neural circuits hold promise for dramatically improving our understanding of brain function. Here I briefly summarize essential genetic techniques to investigate neural circuits along with currently available molecular tools  have been used to visualize neuronal structures both in live and fixed tissues . Multi-c(egfp) gene. Since RG is involved in the assembly of viral particles and the trans-synaptic transportation of the viruses, recombinant RVs cannot spread to presynaptic input cells without exogenously supplied RG. Therefore, when starter neurons express RG, recombinant RVs can spread only once to presynaptic input cells but are unable to spread beyond direct input cells. To achieve selective infection in defined starter cells, recombinant RVs are pseudotyped with the envelope glycoprotein (EnvA) of an avian virus. Pseudotyped recombinant RVs specifically infect starter cells expressing the EnvA receptor, TVA. Thus, when starter cells express both RG and TVA, and pseudotyped recombinant RVs infect these cells, direct input neurons are labeled with EGFP. The drawbacks of this method are (1) underestimated numbers of input neurons due to incomplete labeling caused by unknown reasons, and (2) toxicity that is observed approximately 10 days after infection. Nevertheless, this viral strategy is currently the most powerful approach to identify connected neurons at the light microscopy level.Gaining a wiring diagram of the brain helps to predict and understand how the brain works. Traditional methods such as electron microscopy, paired recordings, and dye-filling have pioneered this field  but thesCaenorhabditis elegans, and is called GFP reconstitution across synaptic partners  between fluorescent proteins. Prototypic genetically encoded voltage indicators were constructed simply by fusing a fluorescent protein with a voltage-sensing domain of an ion channel , consisting of calmodulin (CaM), which contains a calcium-binding site, the M13 peptide, which binds to the calcium-bound form of CaM, and a circulary permuted EGFP  and yellow fluorescent protein (YFP) linked by a flexible 24 amino acid linker . WithoutNeuromodulation has a significant impact on neuronal processing in the normal brain and has been implicated in the pathogenesis of several psychiatric diseases . In partRapid bidirectional manipulations of neural activity are required for providing evidence for the causal relationship between spike patterns and behavior/brain function. Recent developments in optogenetic tools has enabled optical control of neural circuits by driving or inhibiting neuronal spikes with light. Channelrhodopsin-2 (ChR2) is a non-selective cation channel that is opened by blue light . When exN-oxide  Chronic stimulation or inactivation can be easily achieved in DREADD systems by drug application while chronic photostimulation of optogenetic tools in live animals may not be practical due to necessity of long-lasting anesthesia. (3) Neurons in deep brain tissues can be manipulated in DREADD approaches while it is not trivial to deliver light to deep brain areas.Another approach to enhance and reduce neuronal activity is pharmacogenetic tools such as designer receptors exclusively activated by designer drugs (DREADDs). DREADDs are genetically engineered muscarinic acetylcholine receptors, which are insensitive to endogenous acetylcholine but sensitive to the synthetic ligand, clozapine-ide CNO; . DREADDschannels . Advantachannels . These fOne of main challenges in neuroscience is being able to reveal gene expression profiles that underlie the assembly, organization, and function of a defined neuronal population. Genetic materials can be purified from fluorescently labeled target cells collected through physical methods such as laser-capture microdissection, fluorescence-activated cell sorting, and manual sorting. However, since these procedures cause physical damage and stress, the physiological condition and normal gene expression of cells can be disrupted when isolating cells of interest. Recent advances in genetic tagging methods overcome this obstacle and provide a way to obtain “intact” gene expression profiles in a select neuronal population. The translating ribosome affinity purification (TRAP) method uses EGFP-tagged ribosomal protein L10a to pull down mRNAs in a polysomal fraction with anti-GFP antibodies . The RibOther useful genetically encoded biosensors include ones that report synaptic events such as synaptic vesicle fusion and intracellular signaling in dendritic spines. The superecliptic pHluorin (SEP) is a pH-sensitive GFP whose fluorescence is quenched by the acidic condition within synaptic vesicles or endosomes and increases as the pH goes up . The synThe most remarkable feature of GABAergic interneurons is the diversity in cell types, which is a basis of various inhibitory regulations in neuronal circuits . TherefoGABAergic interneurons have been classified by several definitions such as axonal and dendritic morphology, connectivity, electrophysiological characteristics, gene expression pattern, developmental origins and features, and physiological function at a circuit level . These cGenetic targeting is probably the best approach to precisely and reliably identify and manipulate specific cell types. Along with the recent development of genetically encoded molecules such as FRET probes and optogenetic tools, genetic targeting methods have become more efficient and accurate, which dramatically promotes the investigation of the mysteries of neural circuits. Here, I will summarize several genetic methods that can be used to target select neuronal populations and introduce a project to systematically generate Cre driver lines that target subtypes and progenitors of cortical GABAergic interneurons.cis-regulatory elements , or the recombinant bacterial artificial chromosome  containing an exogenous gene and a nearly complete set of cis-regulatory elements  is randomly integrated into the host genome. The advantage of this strategy is that the process from vector construction to mouse generation is relatively straightforward and higher expression of an exogenous gene is expected as multiple copies of transgenes tend to be tandemly integrated into the genome. On the other hand, the transgenic method includes inherent disadvantages; the expression of a transgene may not completely recapitulate the expression pattern of the endogenous gene and may vary among transgenic mouse lines, which demands countless hours of screening to find the appropriate lines. Such incompleteness can be explained based on mechanisms for mammalian gene expression. First, coordinated activity of multiple cis-regulatory elements, which are distributed in the genome occasionally far away from the transcription start site, are essential to drive proper gene expression  containing a gene of interest and minimal pression . Transgepression . The compression . Althougpression . These lFigure 3C). Unlike transgenic methods, gene targeting is the most precise and reliable strategy used to express transgenes faithfully following the expression pattern of the target gene because an exogenous gene of interest is inserted at an endogenous genomic locus with nearly intact gene regulatory environment. Recent advances in the fidelity of PCR enzyme, BAC recombineering technology . The transgene shows a distinctive expression pattern depending on the insertion site that is affected by a unique set of enhancers. The most successful example using this strategy is the GAL4 (the yeast TF)/UAS (GAL4-binding upstream activation sequence) system in fruit flies . The tTA has been modified to generate the rtTA, which is active only in the presence of tetracycline . Thus, in this binary expression system, the level, density, and timing of the transgene expression can be controlled, choosing the amount and administration timing of tetracycline. In general, systems relying on transactivation are reversible, which may be advantageous for some purposes.In transactivation-based systems, distinct driver lines express transcriptional activators in different patterns and responder lines include genes of interest preceded by promoter sequences that bind activators. The advantage of this system is that transcriptional amplification can increase the level of the transgene expression. One of the most common transactivation-based systems is the GAL4/UAS system, which has been frequently used in fruit flies but not in mice . In micecis-regulatory elements and responder lines containing a ubiquitous strong promoter and a gene of interest separated by a transcription stop cassette flanked by two recombinase recognition sites  and the Flp/frt system are two major representatives of binary recombination-based systems in mice  and FlpER . This has been particularly useful in studying whole developmental events of certain temporal cohorts of neurons. Besides temporal control, inducible Cre and Flp can be activated only in a small number of cells within a given population, with lower amount of tamoxifen, which enables experiments at a single cell level . This method requires three genetic components; (1) Cre/CreER driver lines expressing Cre/CreER under the control of cis-regulately elements of gene A, (2) Flp driver lines that express Flp following the expression pattern of the endogenous gene B, and (3) dual responder lines containing a gene of interest and dual STOP cassettes with loxP and frt sites. When these three alleles are brought together, by breeding animals, the exogenous gene of interest is turned on only in cells that have expressed Cre/CreER and Flp sequentially or simultaneously, where dual STOP cassettes are removed. Therefore, the intersectional approach is promising for labeling and manipulation of more specific and narrower populations defined by combinations of neurochemical and developmental features.More specific cell types can be defined by expression of two or more genes. For instance, at least some Martinotti cells are delineated by expression of SOM and CR . A selecFigure 5A); (2) terminal differentiation markers expressed in all (gad2) or broad subtypes of mature and/or developing GABAergic interneurons . In the TF lines, CreER was inserted at the translation initiation codon of the target gene locus to achieve the maximal expression level . In the terminal differentiation marker lines, Cre was integrated immediately after the translation STOP codon followed by the IRES sequence as well as at the start codon of the target endogenous gene . The extensive characterization of these knockin lines by crossing with Cre-dependent GFP and RFP reporters have demonstrated that in almost all cases recombination patterns faithfully recapitulate those of endogenous gene expression. For instance, in gad2-ires-Cre lines, more than 90% of cells expressing GFP induced by Cre activity are Gad67 (a pan inhibitory interneuron marker) positive and the fraction of Gad67 positive cells expressing GFP is more than 90%, indicating a high degree of specificity and efficiency. Inducible Cre lines also show high specificity and reasonable efficiency. In gad2–CreER lines, the density of reporter expression can be adjusted by tamoxifen dosage and almost all major interneuron subtypes are captured, as indicated by coexpression with PV, SOM, CR, VIP, and nNOS. It is also shown that other Cre drivers successfully target virtually all non-overlapping broad subtypes of cortical interneurons, such as SOM, VIP, and CCK, with great precision and efficiency.Recently, systematic efforts supported by the NIH Blueprint for Neuroscience Research have been made to generate and characterize nearly 20 Cre or CreER knockin driver lines targeting GABAergic mature neurons and embryonic progenitors . In thisIn SOM-ires-Cre drivers, major dendrite-targeting interneuron subclasses are captured in the cortex and hippocampus. In the cortex, the dense axonal band of Martinotti cells that target apical tufts of pyramidal cell dendrites is visualized in L1. In the hippocampus, the prominent axonal band of O-LM cells that form synaptic connections with apical tufts of CA1 pyramidal neurons is labeled in the stratum lacunosum moleculare. SOM starts to be expressed in developing interneurons, which just exit the SVZ of the MGE, from mid-gestation onward . ConsistVIP-ires-Cre driver lines specifically and efficiently capture VIP-expressing neurons that are derived from the CGE. In these animals, typical VIP neurons that extend vertically oriented dendrites and axons are visualized. Unlike SOM neurons, VIP neurons show no axonal arborization in L1 in the cortex. In the hippocampus, VIP neurons display two distinct axonal bands in stratum pyramidale and stratum oriens. Electrophysiological studies indicated that neurons labeled in this line show unique intrinsic properties similar to those observed in rat VIP neurons, confirming specificity from the physiological side. VIP expression can be seen from the neonatal stage and developing VIP neurons are also labeled in the immature cortex of VIP-ires-Cre drivers. Thus, VIP-ires-Cre lines are useful for studying development and function of CGE-derived interneurons.CCK-expressing neurons comprise one class of basket interneurons that innervate the soma and proximal dendrites of pyramidal neurons. When CCK-ires-Cre drivers are simply combined with Cre-dependent reporters, no selective labeling of CCK-expressing neurons occurs, perhaps because CCK is not only expressed in GABAergic interneurons but also pyramidal neurons. To overcome this limitation, the intersectional method is applied to target GABAergic CCK-expressing neurons in the cortex. Dlx5/6-Flp is a transgenic line expressing the Flp in most cortical GABAergic interneurons. Combination of CCK-ires-Cre, Dlx5/6-Flp and FSF-LSL-GFP can specifically visualize GABAergic CCK-expressing neurons. In the cortex, CCK and PV (the other class of basket interneurons) terminals are differentially labeled around the same pyramidal neuron. Therefore, the intersectional method provides genetic access to GABAergic CCK-expressing neurons, facilitating studies of their migration, circuit integration, and function.Genetic fate mapping using TF lines that express CreER in MGE progenitors has been used to study how lineage and birth timing contribute to specification of interneuron subtypes and the subsequent migration, circuit integration and functional emergence of postmitotic interneurons . The NkxThis first round effort to generate Cre driver lines targeting GABAergic interneurons has made it possible to dissect diverse and complex cortical GABAergic circuits at a much finer level and has changed the experimental approach used to study the GABAergic system. It has been proven that in combination with recent innovation of genetically encoded molecular tools, these lines are extremely instrumental in studying connectivity, circuit and system level function, and origin and development, of cortical interneuron subtypes . Future One of the most striking features in the GABAergic system is that distinct subtypes form synaptic connections onto different subcellular compartments of excitatory pyramidal neurons . Among vin vivo recordings provided results implying an inhibitory role of ChCs in control of neural circuit activity. It has been shown that hippocampal ChCs spike in antiphase to pyramidal neurons during theta wave oscillation, and fire right before pyramidal neuron spiking during sharp wave associated ripples . Reproducible labeling of spatially and temporally defined ChC progenitors in the VGZ enabled to not only examine whole developmental events but also to study laminar and area distribution of a group of ChCs in the adult cortex . The results have shown that ChC neuroblasts migrate with specific routes and schedules, and settle in upper L2 and L5/6 of the cortex. These ChCs are significantly more enriched in the frontal cortex including the cingulate, prelimbic, and infralimbic cortices compared to the sensory cortex, such as the visual and auditory cortices. Immunohistochemical analysis demonstrated that only a subset of ChCs captured by this genetic fate mapping technique is immunoreactive for PV, which is contrary to the generally believed idea that ChCs are PV-expressing neurons. In addition to uncovering embryonic origin and fundamental organization throughout the cortex, of ChCs, importantly, this study established reliable genetic access to ChCs, which allows targeted introduction of genetically encoded tools in ChCs. Future experiments taking advantage of targeted manipulations in ChCs are expected to address the remaining critical questions, including the following issues. (1) Molecular mechanisms that determine ChC identity are totally unknown. Candidate genes need to be identified and functionally tested in ChC progenitors. (2) Cellular and molecular mechanisms that enable ChCs to establish characteristic axonal arbors and synaptic specificity at AISs are totally unknown. The first step will be to describe the whole process of axonal development at a single cell level. (3) Although the laser scanning photostimulation technique suggested that L2/3 ChCs receives excitatory inputs from L2/3 and 5a, and inhibitory inputs from L1 and L2/3 , an Nkx2.1-expressing remnant of the MGE, during late gestation (and L2/3 , cell tyand L2/3 . StructuThe extreme complexity of cortical circuits has been a huge obstacle to gaining a precise understanding of how the brain is constructed and operates. It is obvious that the diversity of cortical GABAergic interneurons in their morphological, physiological, histochemical, and anatomical properties contributes to the structural and functional complexity of cortical networks. Thus, dissecting specification, development, connectivity, and function of each GABAergic interneuron subtype will be a key topic of investigation to understand cortical function. Simultaneous development of genetically encoded molecular tools and genetic targeting of GABAergic interneuron subtypes has been critical to address these questions. In particular, Cre/CreER driver mouse lines targeting embryonic progenitors and major subtypes of mature GABAergic interneurons have provided the most powerful and versatile strategy to interrogate cell type specific issues. Although we have just obtained genetic access to broad subtypes of GABAergic interneurons, more distinct and homogenous cell types such as NGFCs or Martinotti cells has never been captured by a current set of Cre/CreER drivers, except for ChCs targeted by use of Nkx2.1-CreER lines. Gene expression profiling of known subtypes, intersectional strategies using different Cre and Flp drivers, and analysis of temporal, spatial, and lineage mechanisms to diversify cortical interneurons will innovate additional methods to capture such smaller and purer populations. ChCs will become a pioneer model to study development and function of a pure cell type. Abnormal operation of GABAergic circuits has been implicated in many brain disorders such as epilepsy, schizophrenia, and autism. Systematic analysis of each cortical interneuron subtype in disease model mice will elucidate cellular and molecular pathogenesis, leading to innovation of novel therapeutic strategies. Together, genetic targeting of GABAergic interneuron subtypes will break down complex circuit organization into relatively simple and homogenous units and help to reveal the principle of cortical organization and function.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


These extracellular vesicles comprise a heterogeneous group of vesicles of different sizes and intracellular origins. They are described in literature as exosomes if they originate from multi-vesicular bodies, or as microvesicles when formed by a one-step budding process directly from the plasma membrane. Extracellular vesicles contain a multitude of bioactive molecules that are implicated in intercellular communication and in different biological and pathophysiological processes. Mature red blood cells release in principle only microvesicles. In hereditary hemolytic anemias, the underlying molecular defect affects and determines red blood cell vesiculation, resulting in shedding microvesicles of different compositions and concentrations. Despite extensive research into red blood cell biochemistry and physiology, little is known about red cell deformability and vesiculation in hereditary hemolytic anemias, and the associated pathophysiological role is incompletely assessed. In this review, we discuss recent progress in understanding extracellular vesicles biology, with focus on red blood cell vesiculation. Also, we review recent scientific findings on the molecular defects of hereditary hemolytic anemias, and their correlation with red blood cell deformability and vesiculation. Integrating bio-analytical findings on abnormalities of red blood cells and their microvesicles will be critical for a better understanding of the pathophysiology of hereditary hemolytic anemias.Hereditary hemolytic anemia encompasses a heterogeneous group of anemias characterized by decreased red blood cell survival because of inherited membrane, enzyme, or hemoglobin disorders. Affected red blood cells are more fragile, less deformable, and more susceptible to shear stress and oxidative damage, and show increased vesiculation. Red blood cells, as essentially all cells, constitutively release phospholipid extracellular vesicles  These functions dictate the unusual capacity of RBCs to pass through all types of vessels, and even to squeeze in capillaries of smaller diameters than RBCs themselves  are the most abundant cell type in human blood and they function to transport oxygen (Owww.enerca.org) and the portal for rare diseases and orphan drugs (www.orpha.net) are recommended.Hereditary spherocytosis (HS) is the most common type of hereditary hemolytic anemia. The estimated prevalence of this membrane disorder is 1 in 5000 in the white population of Northern Europe. Red blood cell glucose-6-phosphate-dehydrogenase (G6PD) deficiency is the most common enzyme disorder worldwide, affecting 420 million of the world population. Less common is hemolytic anemia due to pyruvate kinase (PK) deficiency with an estimated prevalence of 1 in 20,000 in the white population. Sickle cell anemia is the conspicuous example of anemia due to a hemoglobinopathy. Its prevalence is 1–5 in 10,000. For more prevalence data and other hereditary hemolytic anemias, the ENERCA  website , and tethered to the cytoskeleton network composed of α- and β-Spectrin and actin via protein 4.1R, protein 4.2, and ankyrin Figure . BesidesIn the resting state, RBC membrane phospholipids are asymmetrically distributed across the bilayer, meaning that the neutral choline-containing phospholipids, like phosphatidyl-choline and sphingomyelin, reside in the outer leaflet of the RBC while the charged amino-containing phospholipids, like phosphatidylserine (PS), phosphatidylinosirol, and phosphatidyl-ethanolamine, reside in the inner leaflet of the membrane  Promoting coagulation; (2) Acting as an “eat me” signal for macrophages. The procoagulant activity of PS relies on the catalytic activity of PS. PS acts as a binding surface for prothrombinase and other coagulation factors, increasing the transformation rate of prothrombin into thrombin, a major coagulation molecule Lentz, . AlthougP. falciparum could release microvesicles rich in host and parasites proteins and these microvesicles possessed a potent stimulatory effect on cells of the innate immune system –redox homeostasis, and in inflammatory reactions. These microvesicles may have immunosuppressive activities by down-regulating the macrophages that uptake them. Sadallah et al. showed that RBCs-derived microvesicles were taken up by macrophages and this inhibited the release of tumor necrosis factor-α (TNF-α) and interleukin 8 when these macrophage were exposed to zymosan A and lipopolysaccharide (LPS), well-recognized as macrophage stimulators  a transmembrane/cytoskeleton protein; (2) an enzyme needed for a metabolic reaction in RBCs; or (3) hemoglobin. The phenotypes of hereditary hemolytic anemias range from being clinically asymptomatic to severe life threatening conditions. In this section we discuss the molecular bases of these disorders, and highlight their potential relationships with RBC vesiculation.Red blood cell membrane proteins control the mechanical strength, the elastic deformability and the hydration state of RBCs. Defect in one or more of these proteins may lead to RBC membrane instability, increased membrane rigidity, and/or aberrant hydration state of RBCs. These perturbations often are associated with morphological changes and decreased life span of the RBCs.in vivo  is the most common inherited hemolytic anemia due to a membrane defect. Inheritance is mostly autosomal dominant and peripheral blood smears of HS patients show spherocytes (sphere-shaped RBCs). However, spherocytic cells may also be seen in other acquired disorders like autoimmune hemolytic anemias, thermal injuries, and venom poisoning  is another diverse group of inherited RBC membrane disorders that are transmitted in an autosomal dominant manner with exception of pyropoikilocytosis (HPP), a severe form of HE, which is generally transmitted in an autosomal recessive manner. HE is characterized by elliptical RBCs (elliptocytes) on peripheral blood smears. However, elliptocytes may also be seen in thalassemia and megaloblastic anemias. The clinical manifestations of HE and HPP vary from silent asymptotic conditions to a severe life threatening hemolytic anemia Nagel, .The hallmark of HE is the loss of mechanical stability of the RBC membrane due to defects in membrane proteins maintaining the lateral linkages of the RBC cytoskeleton. These defects include quantitative and/or qualitative defects in proteins α-spectrin, β-spectrin, and protein 4.1. The quantitative defects result from reduced expression of genes encoding for normal proteins. On the other hand, qualitative defects result in an alteration in the protein structure that affects the protein interaction with the adjacent proteins. HE RBCs are more susceptible to shear-stress induced fragmentation which is associated with membrane loss and progressive transformations of RBCs from the biconcave morphology to the elliptical or oval one. Membrane loss is potentially through shear-induced vesiculation, and such loss may be involved in decreased deformability of HE RBCs. However, the exact relationship between HE and vesiculation remains to be studied  RBCs in peripheral blood smears. Like other RBC membrane disorders, the phenotype of HSt patients may range from being asymptomatic to suffering from a severe hemolytic anemia. Membrane leakiness of monovalent cations alters the osmotic pressure across the RBCs membrane, and hence affects RBC volume and cellular deformability. Such changes may decrease the life span of defective RBCs and increase their splenic sequestration  is a genetic disorder that comprises a heterogeneous group of syndromes, in which the RBC membrane is leaky to monovalent cations, showing increased permeability to Nan Bruce, .+, results in a reduction of total intracellular cation content. This induces RBC dehydration associated with elevated MCHC, and thus perturbs RBC deformability  and dehydrated DHSt . In OHSt, RBCs have high intracellular sodium concentration, which results in cell overhydration, reduction in the mean cell hemoglobin concentration (MCHC), and transformation of RBCs into spherotic non-deformable cells. In xerocytosis, increased membrane leakiness, in particular K+/Na+ transport across the membrane, have previously been found. However, Stewart and Turner reported that stomatin knockout mice did not have hemolytic anemia, which may indicate that stomatin deficiency in OHSt is a secondary event and not the primary underlying molecular defect of DHSt . The generated ATP is vital for RBC biological functions, including glycolysis itself, and cellular structures. For instance, ATP is essential for kinase reactions needed for cytoskeleton protein phosphorylation, which is crucial for the integrity and deformability of RBCs  to the reduced form (GSH) and thus protecting critical structures like membrane lipids and cytoskeleton proteins from oxidation. Under high oxidative stress conditions, the pentose phosphate pathway consumes more glucose to compensate for the depleted GSH  deficiency and PK deficiency . G6PD deficiency can be categorized based on the severity into three main variants: (1) Variants associated with episodic or acute hemolytic anemias; (2) Variants associated with chronic hemolytic anemias; (3) Variants associated with no clear risk of hemolysis  are by-products of normal physiological intracellular reactions. However, ROS levels significantly rise upon exposure to exogenous factors like ingestion of drugs with high redox potentials, ingestion of fava beans, and during infections. Under normal conditions, oxidative damage is reversed by adequate levels of GSH. However, G6PD deficient RBCs have low levels of GSH, and they are more susceptible to damaging effects of the accumulated ROS resulting in hemolysis  deficiency is the most common glycolytic enzyme defect and a relatively common cause of hereditary non-spherocytic hemolytic anemias. Patients have variable degrees of hemolysis, leading to mild compensated anemia, moderate hemolysis that exacerbates during infection, to severe transfusion-dependant chronic hemolytic anemia. The disease is due to missense mutations in the Hemoglobin is the main oxygen carrier in humans. It is a tetrameric protein composed of two pairs of different globin chains. Normal adult hemoglobin is mainly comprised of Hemoglobin A (Hb A), which is composed of two α-globin and two β-globin chains. Hemoglobin concentration (32–35 g per 100 mL of packed RBCs), the integrity and the solubility of these hemoglobin molecules, and their oxygen affinity are crucial factors for preserving the RBC's main function, oxygen delivery. More than 1000 mutations in globin-encoding genes have been found to affect hemoglobin synthesis, solubility, stability, or oxygen affinity. Such inherited hemoglobin anomalies may perturb the intracellular viscosity of RBCs and cell deformability, increasing the splenic sequestration of poorly deformable cells. Moreover, these perturbations may trigger proteolytic reactions, associated with ROS release, which may ultimately result in hemolytic anemias Kuypers, .Hemoglobin anomalies can be associated with quantitative or qualitative abnormalities. Qualitative anomalies result from structurally altered hemoglobin, which has perturbed physical and/or chemical properties. Quantitative hemoglobinopathies are due to a defective production of otherwise normal globin chains. Examples of pathological conditions pertaining to the first group are sickle cell anemia, hereditary met-hemoglobinemia, high oxygen affinity polycythemia, and low oxygen affinity cyanosis. Examples of pathological conditions related to the quantitative anomalies are α- and β- thalassemias. By far the most prevalent and important conditions are sickle cell disease and the thalassemia  gene are asymptomatic (sickle cell trait). People who have homozygosity for the mutation that causes HbS suffer from sickle cell disease. Sickle hemoglobin (HbS) polymerizes and undergoes transformation from the soluble state to a highly viscous semisolid gel under low oxygen tension conditions  phenotypes, and disease processes. Such profiling of hereditary hemolytic anemias can be started by integrating data generated by robust methods like ektacytometry, flow cytometry, nanoparticle characterization and proteomics.Ektacytometry is defined in the dictionary of cell and molecular biology as a “Method in which cells  are exposed to increasing shear-stress and the laser diffraction pattern through the suspension is recorded; it goes from circular to elliptical as shear increases. From these measurements, a deformability index for the cells can be derived.” Lackie, . DetermiFlow cytometry is a simple, reproducible, and high throughput method that can be used for qualitative and quantitative immunophenotyping of RBCs. The availability of a wide array of fluorescently labeled antibodies can be utilized to detect several RBC surface and cytoplasmic antigens that may vary among hereditary hemolytic anemias. Additionally, laser scattering at different angles can distinguish differences in cell size and internal complexity  and composition of microvesicles  selection of microvesicles based on a size criterion followed by analysis of the fluorescent signals of events limited to this preselected size, or (2) initial selection is based on the fluorescent signals then limiting the analysis by scatter gating to these signals  is a powerful, automated and high throughput tool, which enables us to analyse the proteome of RBCs in health and disease. Such a comparative proteomic analysis can provide us with considerable insights into disease-related alterations and severity, potential diagnostic markers, potential therapeutic targets, and drug-induced changes (Prudent et al., There is a growing interest in using proteomic technologies for high throughput protein profiling of extracellular vesicles. Such an interest is driven by new findings pointing to the crucial roles in intercellular communications and other pathophysiological processes. With respect to RBC-derived microvesicles, little is known about their protein cargo (Simpson et al., Red blood cells-derived microvesicles, once thought of as cell debris, now seem to have important roles in regulating blood homeostasis and modulating immune response and other pathophysiological processes. Red blood cell microvesiculation is a tightly controlled process and sorting of bioactive molecules into microvesicles occurs in a selective manner. The molecular defects of hereditary hemolytic anemia are diverse, and they potentially affect RBC vesiculation. Microvesicles released in the different hereditary hemolytic anemias are potentially unequal and may have different biological effects. As the exact role of red cell derived miscrovesicles in many of the hemolytic anemias is currently unknown and unexplored this opens up a whole area of exciting research in the coming period of time. Understanding the molecular bases of hereditary hemolytic anemias, and their impact on RBCs deformability and microvesiculation, can provide new insights into the pathophysiology of these anemias and may lead to the discovery of new diagnostic markers and therapeutic molecules.Amr Alaarg, Raymond M. Schiffelers, Wouter W. van Solinge, and Richard van Wijk wrote the manuscript. All authors approved the final version 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.


Arabidopsis thaliana, Zea mays, Orza sativa, and Populus trichocarpa) in a display that facilitates comparisons among orthologs and extrapolation of annotations among species. A single-page view collates key functional data for members of each Putative Orthologous Group (POG): graphical representations of InterPro domains, predicted and established intracellular locations, and imported gene descriptions. The display incorporates POGs predicted by two different algorithms as well as gene trees, allowing users to evaluate the validity of POG memberships. The web interface provides ready access to sequences and alignments of POG members, as well as sequences, alignments, and domain architectures of closely-related paralogs. A simple and flexible search interface permits queries by BLAST and by any combination of gene identifier, keywords, domain names, InterPro identifiers, and intracellular location. The concurrent display of domain architectures for orthologous proteins highlights errors in gene models and false-negatives in domain predictions. The POGs2 layout is also useful for exploring candidate genes identified by transposon tagging, QTL mapping, map-based cloning, and proteomics, and for navigating between orthologous groups that belong to the same gene family.The Putative orthologous Groups 2 Database (POGs2) (http://pogs.uoregon.edu/) integrates information about the inferred proteomes of four plant species ( The ability to acquire genome sequence data far outpaces the ability to infer gene functions. The vast majority of functional data comes from work with a handful of intensively studied model organisms, each of which is represented by a species-specific database that compiles functional data in the context of predicted and validated gene models. For genes that have not been studied directly, extrapolation of functional data from orthologous genes is a powerful method for inferring function, and comparisons of orthologous gene models can make errors in gene models apparent. The information for such comparisons can be extracted from existing species-specific and phylogenetic resources –6, but tArabidopsis thaliana (Arabidopsis) and Oryza sativa (rice), the two plant species whose complete genome sequences were available at that time. POGsDB collated data from multiple resources into a single-page format that allowed users to view salient functional information for orthologous proteins in rice and Arabidopsis at a glance. Here, we describe a major update, POGs2. POGs2 retains the user-friendly single-page view and flexible search strategies of the original POGsDB, but incorporates two additional plant species [Zea mays (maize) and Populus trichocarpa (poplar)], two independent sets of ortholog predictions, and numerous interface improvements as described below. In 2007, we developed the Putative Orthologous Groups (POGs) database to meet this need for plant genomes [The POGs2 database is a MySQL database that organizes information about the inferred proteomes of Arabidopsis, rice, maize, and poplar in the context of Putative Orthologous Groups (POGs). Because large-scale orthology prediction is a developing art , we impoArabidopsis thaliana genome v10, ftp://ftp.arabidopsis.org/; Oryza sativa genome v7, ftp://ftp.plantbiology.msu.edu; Zea mays genome B73 Release 5b (http://ftp.maizesequence.org); Populus trichocarpa genome v8 (ftp://ftp.jgi-psf.org/pub/compgen/phytozome/v8.0/Ptrichocarpa/). Experimentally-validated intracellular locations of maize and Arabidopsis proteins were imported from the Plant Proteome Database (http://ppdb.tc.cornell.edu/) [Other data in the POGs2 database was drawn from the following sources. Inferred proteomes with associated gene descriptions and InterPro domains  were impl.edu/) . InterPrBy collaborating closely with experimentalists whose research requires frequent access to this type of data, we developed a user-friendly interface for searches, data visualization, and navigation to complementary web resources. A single-page format allows users to quickly assess key functional and phylogenetic features of POG members without the need to navigate to multiple pages  and 2. EBecause the validity of the ortholog predictions is essential to support valid cross-species inference, two tools are provided to help users assess the quality of the predictions. First, a statement at the top of each POG-view page indicates whether the Gramene and PLAZA predictions are in agreement ; if not,Buttons on each POG-view page allow users to view or download sequences and multiple sequence alignments of POG members , top, ashttp://angularjs.org) to retrieve JSON data files from our PHP API server, and render them into text and graphics By using this method, POGs2 relies largely on the web browser for rendering data rather than relying on the server alone. Thus, the load time is very quick and the server may handle numerous requests simultaneously without a significant decrease in speed.POGs2 employs a client-side JavaScript library AngularJS (http://redis.io/). POGs2 offers flexible and intuitive options for queries based on gene identifier, keywords, domain names, InterPro domain identifiers, and predicted or established intracellular locations . The keyPOGs2 complements existing database resources for plant genomics by collating a subset of data types available elsewhere in a manner that simplifies protein-centric cross-species functional inferences. POGs2 is distinct in the types of data that are displayed concurrently for orthologous proteins , in the ease of accessing protein sequences and multiple sequence alignments for orthologous proteins, and in the types of searches that are readily accomplished (e.g. searches for orthologous groups based on both conserved domains and intracellular location). Whereas other resources provide greater depth of information about particular species or greater breadth of phylogenetic data, POGs2 offers a rapid first stop for researchers wishing to glean the essence of what is known about the functions of a particular protein and its orthologs. For this reason, the display is limited to four model plant species, and priority was placed on devising a highly responsive interface that minimizes the need to navigate to multiple pages. Links are provided to species-specific and phylogenetic resources for those wishing to explore more deeply or broadly. POGs2 is particularly useful as the starting point for evaluating and prioritizing candidate genes that arise through genetic, coexpression, or proteomic analyses. QTL mapping, transposon-tagging, and map-based cloning approaches for identifying genetic variations that underlie phenotypes typically yield multiple candidates. Likewise, coexpression and coimmunoprecipitation data yield sets of genes that are candidates for harboring functions of interest. It is typically both time-consuming and costly to follow up all such candidates, so informed prioritization is a worthwhile first step. By bringing together gene descriptions, domain architectures, and intracellular location data for orthologous proteins in a single page view, and by providing ready link access to coexpression and publication data in Arabidopsis, users working in both “model” and “non-model” plant species can quickly distinguish the more promising from the less promising candidates. This aspect has been enormously useful in our own efforts to characterize the gene complement underlying chloroplast biogenesis via a large-scale forward genetic approach in maize . In factOther benefits of the POGs2 layout include the ease of access to protein sequences and multiple sequence alignments, and graphics that display domain architectures in a manner that highlights likely errors in gene models and false-negatives in domain predictions see . In the


More than 600,000 head and neck cancers (HNC) are diagnosed every year and the incidence and mortality rates vary according to the economical status of the country. Most are oral, pharyngeal or laryngeal squamous cell carcinomas. The most significant risk factors include lifestyle habits . More recently, exposure to some biological agents (HPV and EBV) and genetic susceptibility were also identified. Tobacco smoking increases acetaldehyde burden following alcohol consumption, and alcohol consumption enhances the activation of pro-carcinogens by induction of cytochrome P450-2E1 system. HPV and EBV are associated with the oropharyngeal and nasopharyngeal cancers, respectively. Genetic susceptibility has a significant role in non-smoking young patients and it is based on differences in the efficiencies of metabolizing carcinogens, DNA repair and cell cycle control. Familial aggregation of HNSCC has been recently reported and the responsible genes are under investigation. Possibly, the identification of a genetic risk profile may improve prevention, early diagnosis and treatment. Most HNC are characterized by local tumor invasion, frequent regional lymph nodes metastasis, and multiple primary cancers in the upper aerodigestive tract, esophagus and lung. Most patients had advanced-stage disease at diagnosis and current treatment options frequently incur significant morbidity. The significant prognostic factors in HNC patients are TNM stage, tumor site and histologic variables. However, tumors of the same site with similar histology and stage may behave differently due to their differing biological characteristics. There is a major interest in the identification of biomarkers  that could be used in the clinical practice in order to better prognosticate and risk-stratify patients and predict treatment response. The results of these investigations can completely change clinical practice and result in effective “personalized medicine”.Only HPV positivity and EGFR overexpression are currently considered reliable predictive markers for therapeutic response in HNC patients. Some recently published studies have also shown that TP53 genetic polymorphisms and mutations influence tumor progression and response to therapy. Genotyping has being also utilized to probe other known gene mutations and can offer potential for identification and targeting of key pathways implicated in tumor progression to guide therapeutic strategies. Recently we have investigated the expression profile of Epithelial-Mesenchymal-Transition genes in patients with oral SCC using a cDNA microarray platform coupled to qRT-PCR and immunohistochemical analysis. We identified TWIST1 transcription factor to be overexpressed and to predict metastases and prognosis. Most prognostic information on HNC are patient and tumor-related factors and occasionally pathologic findings. These variables influence outcomes prediction at diagnosis, but do not consider several others such as response to initial therapy, complications, risk of recurrences and second primary tumors. Furthermore, the risks of recurrence, second primary cancers and non-cancer related deaths change during follow-up when a patient develops a recurrence or second primary tumor. More sophisticated prognostic evaluations are ongoing.There are no competing interests in this presentation.


Alexander Tsai and colleagues highlight the complex relationship between poverty and HIV stigma in sub-Saharan Africa, and discuss possible ways to break the cycle.Please see later in the article for the Editors' Summary Poverty is an important driver of HIV stigma.HIV-related morbidity undermines HIV-infected persons' ability to maintain their full economic contributions to family and community life.Stigmatization and exclusion from local solidarity networks erode food and livelihood security, which undermine adherence to HIV treatment, further perpetuating this cycle.HIV treatment can improve the economic, mental, and social health of HIV-infected persons, but it may not be sufficient to reduce the stigma of HIV.Livelihood interventions can reduce the stigma of HIV by directly targeting poverty.HIV is highly stigmatized throughout sub-Saharan Africa Given the pressing need for effective intervention, in this Policy Forum article we argue that poverty alleviation should be conceptualized as a potentially powerful yet understudied tool for confronting the stigma of HIV. Beginning with a conceptual discussion of stigma, we draw on qualitative and epidemiological studies to focus our attention on the perceived disability, economic incapacity, and death associated with HIV as key determinants of stigma in numerous sub-Saharan African countries. We then explain how poverty alleviation may help reduce the stigma of HIV. In doing so we borrow from disparate lines of inquiry, including HIV research and programmatic work in sub-Saharan Africa; the medical literature on other stigmatized conditions, such as epilepsy and schizophrenia; and literature from other fields, including anthropology, economics, psychology, and sociology.In many cultural contexts, the stigma of HIV is largely driven by concerns about its symbolic meanings Goffman Persons with and without the stigmatized attribute are separated into two categories: “them” vs. “us” Internalized stigma results when stigmatized persons come to accept these inhospitable attitudes as valid, thereby developing self-defacing beliefs and perceptions about themselves Enacted stigma results when clandestine hostility and/or overt acts of discrimination are directed towards persons specifically because they possess the stigmatized attribute felt stigma) In such an environment, even stigmatized persons who are not directly victimized may experience fear in anticipation of being targeted  as well as the stigmatizers .Table S1Poverty and lack of reciprocal exchange as important drivers of stigma, described in qualitative studies conducted in sub-Saharan Africa.(DOCX)Click here for additional data file.Table S2Local dysphemisms for HIV-infected persons.(DOCX)Click here for additional data file.


The detachment of atherothrombotic material from the atherosclerotic coronary plaque and downstream embolisation is an underrecognized phenomenon and it causes different degrees of impairment of the coronary microcirculation. During treatment of obstructive atherosclerotic plaque by percutaneous coronary intervention (PCI) distal embolisation (DE) is considered to be inevitable and it is associated with potential clinical and prognostic implications. This review aims to assess the main aspects of both spontaneous and procedural DE, analyze their different pathophysiology, provide specific insights on the main diagnostic tools for their identification, and finally focus on the main strategies for their treatment and prevention. Atherogenesis is the cause of ischemic heart disease and knowledge about its pathophysiology is progressively increasing. Progression of plaque within a vessel is not merely a consequence of cholesterol accumulation but a dynamic process related to a complex interaction of several risk factors . IntermiTreatment of obstructive atheroma by coronary balloon angioplasty was originally described by Gruentzig in 1977. Since then progressive and remarkable innovations in techniques and materials have made percutaneous coronary intervention (PCI) a cornerstone for the treatment of obstructive coronary artery disease (CAD) in many clinical settings .However, achievement of epicardial coronary artery patency does not always translate into a complete and effective myocardial perfusion . This phIndividual susceptibility, ischemic-reperfusion, injury and distal embolisation (DE) are the main mechanisms leading to NR . Among tThis review article aims to shed light on this complex phenomenon, providing insights about the pathophysiology of DE and the available methods to detect and prevent it.The term “distal embolisation” refers to the detachment of athero or atherothrombotic fragments from the atherosclerotic plaque and their dislodgement downstream the peripheral portion of the coronary tree. This leads to the occlusion of coronary microcirculation with consequent ongoing myocardial ischemia and necrosis. Compared to classical occlusion of an epicardial segment, DE produces patchy microinfarcts in the area at risk , with diDE was described for the first time in human in the 1980s by Falk and Davies in two series of patients with sudden death due to coronary thrombosis . The twoThe interest in DE has been renewed after the development of PCI in which DE is related to balloon dilation or stent deployment. This allows labelling DE as “spontaneous” and “procedural” . Main diSpontaneous DE is a phenomenon typically observed in ACS patients. It is strictly related to the pathophysiology underlying plaque instability, which is a progressive process going on for several days before the abrupt occlusion of the epicardial coronary artery. Late stage thrombi (>7 days), indeed, have been found in 69% of culprit plaques . In thisBroadly, spontaneous DE can be considered to be universal in coronary artery disease (CAD) and is a major component in processes accounting for ACS pathophysiology .μm, a cut-off derived from distal filter studies [In terms of embolisate dimension, both macro- and microembolisation contribute to spontaneous DE. Macroembolisation refers to dislodgement of fragments with a diameter greater than 100  studies , and it  studies .μm. Platelet-platelets aggregates, neutrophils-platelets aggregates, monocytes-platelets aggregates [Microembolisation refers to embolised debris with a diameter below 100 gregates , microthgregates , amorphogregates , micropagregates , cholestgregates , and leugregates . Most ofgregates . Its congregates . This phgregates –23.Procedural DE is strictly related to PCI procedure and it can occur both in stable and ACS patients. It contributes to the pathophysiologic background of postprocedural myocardial infarction (Type 4a) .Procedural DE recognises risk factors including hypertension , diabeteμm, 30% 80–120 μm, 16% 120–250 μm, 15% 250–50 μm, and 17% >500 μm) [Procedural DE composition varies from ACS to stable angina. In the ACS setting, procedural DE components are similar to those of spontaneous DE, mainly consisting of atherothrombotic debris, amorphous hyaline material (macroembolisation), foam cells, cholesterol crystals, platelet-platelet aggregates, leukocytes-platelet aggregates, microparticles, coagulation material (microembolisation) with a high content of tumor necrosis factor alpha, serotonin, thromboxane, and tissue factor  . As for >500 μm) .In stable patients procedural DE consists mainly of fibrotic and hyaline material with few cells  . ConsequClear evidence of procedural DE comes from intracoronary Doppler-flow-wire studies which reported firstly a typical reversal systolic flow with a delayed diastolic component and secondly a direct identification of macroemboli appearing as high intensity transient signals on the Doppler spectrum . Furtherμm microspheres. This infusion resulted in left ventricular impairment with inotropic reserve reduction associated with a compensatory increase of basal coronary blood flow [The relationship between myocardium and DE has been assessed in animal models with injection of inert 40 ood flow  due to aood flow . The incood flow .This “contraction-perfusion” mismatch represents a peculiarity of DE.Both autopsy studies and CMR studies , 36 confIt is likely that procedural DE interaction with microvasculature and myocardium is related to the clinical setting. Indeed in stable patients procedural DE, acting on a viable and “healthy” myocardium, causes an injury pattern close to that observed in animal models, thus accounting for the damage to a small myocardial volume (up to 5%). In ACS patients, procedural DE amplifies myocardial injury, acting on a severely ischemic and necrotic area which, after epicardial coronary artery occlusion, has suffered from the phenomenon of ischemia-reperfusion injury and previous spontaneous DE .Isolated spontaneous DE has been clearly described in postmortem studies on patients who died from sudden death due to coronary thrombosis , 10. DifA number of technologies are available to indirectly assess the occurrence of DE, by detecting periprocedural myocardial injury and MVO .Troponins are proteins involved in the regulation of cardiac and skeletal muscle contraction, acting by modulating the interaction between actin and myosin. There are three cardiac troponin isoforms, namely, troponin C , I (inhibitory), and T (tropomyosin binding), and each is involved in a specific step of calcium mediated actin-myosin interaction .Creatine kinase (CK) is an enzyme involved in the regulation of cellular metabolism, catalysing the conversion of creatine to phosphocreatine thus modulating adenosine triphosphate (ATP) intracellular levels. There are three different isoforms, whereas CK MB is the one expressed by myocardium .Increased levels of troponin I or T and of CK MB isoform represent an easy way to detect myocardial injury. Evidence of troponin increase on admission in ACS patients is probably related to spontaneous DE. This is particularly relevant in late presenting patients with older intracoronary thrombi, intermittent epicardial artery occlusion, and showers of microemboli distally to the coronary microcirculation.There is some debate about the relevance of elevation of biochemical markers of myocardial injury after PCI. Troponin is excellent for risk stratification in ACS and has an advantageous release profile. However, it may be oversensitive for assessing postprocedural myocardial injury, especially since the advent of high-sensitivity troponin assays . At highCK MB elevation has a less complex relationship between myocardial injury and myocardial necrosis after PCI . Indeed,Moreover, it must be emphasised that postprocedural myocardial injury marker increase may not be entirely related to DE. Two patterns of postprocedural myocardial damage are described by CMR, namely, “distal” and “peristent.” Distal is directly related to procedural DE occurrence, whilst peristent reflects the occlusion by the stent of small branch vessels .Based on the infusion of small microbubbles able to remain completely within the microvasculature, myocardial contrast echocardiography (MCE) can detect areas of MVO as region of persistent contrast defect in the territory of the reopened culprit vessel . MoreoveHigh spatial and temporal resolution and the relatively low cost are the main advantages of MCE which is however an operator-depending technique . It mustContrast-enhanced CMR represents, at the state of art, the gold standard for MVO assessment, with a good sensitivity, superior to MCE .Two CMR strategies are available to identify MVO. In the first passage method, only after two minutes from contrast injection, MVO appears as hypoenhanced area while the whole myocardium results moderately hyperenhanced. This method is affected by low spatial resolution, reduced left ventricular coverage, and then low diagnostic sensitivity .Higher spatial resolution, increased left ventricular coverage, and the least variability can be obtained with late gadolinium enhanced approach, in which image acquisition, after 10–15 minutes from contrast dye injection, allows detection of MVO as a hypoenhanced area within the hyperenhanced infarct region , 58.Also the timing for CMR scanning has been shown to be relevant since only persistent MVO observed up to one month is really associated with worse regional wall motion and poor prognosis .Notably CMR allows discriminating the mechanism of postprocedural myocardial injury. Indeed, two kinds of late gadolinium enhancement have been described after PCI. A so-called “persistent” pattern, in which the enhanced area is very close to the deployed stent, is caused by poststenting side branch occlusion. In comparison the “distal” pattern is described when the enhanced area is located at least 10 mm downstream from the stented siste and it is caused by procedural DE . Higher Despite cost, availability issues, and difficult performance in unwell patients, high specificity and sensitivity makes CMR the reference technique for the assessment of acute and chronic consequences of DE.Typical electrocardiographic signs of DE have been identified in patients undergoing elective PCI and postprocedural myocardial injury defined as troponin increase . While tMoreover, evaluation of ST resolution at surface ECG has been easily applied to assess the occurrence of MVO and thus indirectly both spontaneous and procedural DE, in STEMI patients undergoing primary PCI .TIMI flow grade  and corrHowever, the main MBG limitation remains its interobserver variability which can be overcome with a new automated MBG quantification software ) developed by Zwolle's group  and valiIntravascular imaging, namely, intravascular ultrasound (IVUS) and optical coherence tomography (OCT), by providing detailed insights into atherosclerotic plaque composition and structure, might be useful in the early identification of prone-to-DE lesions. In this regard, the presence of a necrotic core component derived from virtual histology-IVUS and the morphologic characteristics of plaque derived from grayscale IVUS have been shown to be closely related to procedural DE after PCI , 70.Due to its recent introduction in the clinical arena, there is not enough evidence for OCT role in DE diagnosis. However, an additive predictive value of OCT for post-PCI CK-MB elevation in case of IVUS-echo attenuated plaques  has beenThe adoption of the pressure-thermistor-tipped guidewire might provide relevant details about the condition of coronary microcirculation. In this regard an increased index of microcirculatory resistance (IMR), defined as the ratio between distal pressure and the inverse of the hyperaemic mean transit time, has been recently related to poor long-term outcomes  showing Thus, even if their introduction in the clinical arena still needs confirmation from larger clinical trials, both intravascular imaging and coronary functional tests might find an application as diagnostic tools for DE detection and prevention.Due to the relevant clinical impact of DE, especially in STEMI patients, several strategies, both pharmacological and mechanical, have been developed to prevent or to reduce the detrimental implications of DE. These strategies, mainly applied in ACS patients, are a useful tool to prevent procedural DE and to treat the consequences of spontaneous DE .Pharmacological strategies can be applied to prevent and mainly to limit the myocardial injury in case of spontaneous DE. However, the same pharmacological tools can be adopted to reduce the myocardial damage whenever prevention of procedural DE fails.Three main categories of drugs are available: antiplatelet drugs, statins and vasodilators.(1) Antiplatelet Drugs. By reducing the thrombotic burden all these agents can limit the occurrence and the amount of both spontaneous and procedural DE.A direct effect of aspirin on prevention of DE has never been investigated before. However, recent data support a possible contribution of aspirin in DE prevention by reporting a higher thrombus burden  and a poA direct benefit in terms of periprocedural myonecrosis reduction, and thus in terms of DE prevention, has been shown for P2Y12 receptors antagonists.The reduction in thrombotic endpoints observed in the PCI-CURE , TRITON The strength of antiplatelet activity appears to be dose- related for clopidogrel, as supported by the ARMYDA trials in which higher clopidogrel loading  and mainSimilarly, the analysis of patient-level data from the three CHAMPION trials might support a role for cangrelor, a new fast-acting intravenous antiplatelet agent, in limiting the occurrence and the consequences of DE .Besides P2Y12 receptors antagonists, also GPIIb/IIIa inhibitors may reduce downstream embolisation local generation of thrombus as well vasoactive and chemotactic mediators release from activated platelets .A large core of evidence supports the application of GPIIb/IIIa inhibitors in high-risk PCI with evidence of highly thrombotic burden but a still open debate is about the best timing and the best route for their administration.After the first evidence of major adverse cardiac events (MACE) reduction with preprocedural administration , a clearThe CICERO trial reported a better MBG and a lower postprocedural myonecrosis using intracoronary administration although no differences in the incidence of MACE were found . The INFA local abciximab delivery strategy by adoption of specifically designed catheters has been purposed. The ongoing COCTAIL II trial will specifically address this issue , after t(2) Statins. Statins exert a stabilizing action on atherosclerotic plaque, protecting from coronary events and leading to a reduction of periprocedural myocardial damage occurrence [currence . Statinscurrence , 95. Notcurrence  has also(3) Vasodilators. Since DE causes both mechanical and functional microvascular impairment, vasodilators would act by dilating the obstructed microcirculation, counteracting the vasoconstrictive activity of embolisate. Two main vasodilators agents are currently used in the Cath-Lab to limit the impact of DE: adenosine and sodium nitroprusside.Adenosine causes vasodilation by binding A2 receptors and inducing adenylate cyclase and platelets nitric oxide synthase activity. At the same time, adenosine exerts an anitinflammatory activity by hampering neutrophils accumulation, limiting free radicals production, damping endothelin-1 synthesis, and preventing intracellular Ca++ overload . The firSodium nitroprusside is a potent endothelium independent vasodilator acting directly on vascular muscle cells, providing a longer maximal coronary hyperemia at a lower dose compared to adenosine . In the The low nitroprusside dose adopted in the study and the large number of pleiotropic effects exhibited by adenosine are possible explanations for the superiority of intracoronary adenosine in terms of microvascular protection.Other agents such as nondihydropyridine calcium channel blockers (verapamil and diltiazem), endothelin antagonists, and nicorandil have been purposed to rescue coronary microcirculation from DE, but their use in clinical daily practice is still limited due to lack of strong evidence.Even if a clear advantage in terms of clinical outcomes has not been confirmed in larger clinical randomized trials, the benefit in terms of infarct size reduction and lower occurrence of MVO supports the current vasodilators adoption, especially adenosine, in case of spontaneous and procedural DE. As for GPIIb/IIIa, also for vasodilators, and again mainly for adenosine, there is a debate about the best time (before/during versus after PCI completion) and the best route of infusion (intracoronary versus intravenous) and ongoing studies will soon address this issue.Broadly, most of the studies until now have mainly investigated adenosine use after procedure completion; however, since adenosine has a central role in myocardial conditioning , it is rMore debated is the issue about the best route of administration. Indeed after the first large clinical trials confirming the effectiveness of intravenous route, intracoronary adenosine administration has been shown to be safe and feasible , with a While pharmacologic approaches for prevention and treatment of procedural DE overlap with those of spontaneous DE, mechanical prevention is a strategy specifically addressed to prevent the occurrence of procedural DE.Balloon/filter protection devices and thrombectomy constitute the equipment for interventional cardiologist dealing with PCI in settings of higher risk of DE, namely, thrombotic lesions and degenerated SVG.(1) Balloon/Filter Protection Devices. Balloon/filter devices consist of three main types: distal occlusion devices, distal filters, and proximal occlusion devices. Among them, distal filters, consisting of baskets placed distally to lesion, are the most used in clinical practice. Their adoption is however limited to PCI on SVG, since main clinical randomized trials failed to show a real clinical benefit in PCI on native vessels [ vessels . Embolis vessels , 110. (2) Thrombectomy. Aspiration of thrombus material from the coronary lumen is the principle underlying thrombectomy. By providing thrombus debulking, thrombectomy significantly reduces the risk of procedural DE in highly thrombotic lesions and it could also account for a lower probability of spontaneous DE in case of STEMI patients treated with a “lone thrombus aspiration” approach  [tenting) .This tool for DE prevention has currently a IIa indication according to European guidelines in STEMI patients . After tTwo main kinds of thrombectomy devices are available at the moment: manual and mechanical. In manual thrombectomy devices, thrombus aspiration is obtained by application of a suction force exerted by the operator. In mechanical devices, aspirating force is produced by specific device related mechanisms.Greater profiles, lower flexibility, and a steeper learning curve have been advocated as the main weak points of mechanical devices. Only two randomized trials have directly compared manual versus mechanical aspiration and both reported no differences between the two approaches in terms of MACE, highlighting however a higher rate of successful deployment of the manual thrombectomy device compared to the mechanical one , 115. ThRecently, the interest for manual thrombectomy has progressively increased. After the first studies ascertaining its benefit in terms of myocardial reperfusion , MCEven if not designed to address this issue, the TAPAS was the first large study enrolling up to 1071 STEMI patients, reporting not only a better myocardial reperfusion in thrombectomy group , but alsNCT01149044).These results, however, have been recently debated in the TASTE trial which failed to find significant differences in terms of mortality, recurrent myocardial infarction, and stent thrombosis at thirty-day follow-up between manual thrombectomy group and controls . The stuHowever, the TASTE trial could indirectly suggest that thrombectomy might really make the difference in specific settings featured by a higher risk of DE such as in patients with large thrombotic burden , extendeFinally the results from TASTE trial and TROFI study , which fDE needs to be considered in the pathophysiology of CAD and it represents a key event during PCI. Spontaneous and procedural DE can both lead to myocardial injury but the main clinical implications are in PCI and in STEMI patients. Several diagnostic tools are available to detect myocardial injury following DE and progress in intravascular imaging and functional intracoronary tests will be helpful in early detection of DE occurrence and upstream identification of those anatomical settings at higher risk of DE.Thrombus aspiration, GPIIb/IIIa adoption, dual antiplatelet therapy, and intensive statin treatment are recommended to limit the impact of both spontaneous and procedural DE.