The endocannabinoid system plays a key role in regulating a variety of physiological processes such as appetite control and energy balance, pain perception, and immune responses. Recent studies have implicated the endocannabinoid system in the regulation of bone cell activity and bone remodeling. These studies showed that endogenous cannabinoid ligands, cannabinoid receptors, and the enzymes responsible for ligand synthesis and breakdown all play important roles in bone mass and in the regulation of bone disease. These findings suggest that the endocannabinoid pathway could be of value as a therapeutic target for the prevention and treatment of bone diseases. Here, we review the role of the skeletal endocannabinoid system in the regulation of bone remodeling in health and disease. Endocannabinoids and their receptors are involved in the regulation of numerous physiological processes including neurotransmission, pain perception, learning, memory, cardiovascular homeostasis, appetite, motor function, and the immune response (reviewed in N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG). The endocannabinoids anandamide and 2-AG are responsible for most pharmacological actions associated with cannabinoid receptors in mammalian cells (Reviewed in N-arachidonoyl phosphatidylethanolamine (NAPE) by the enzyme NAPE-phospholipase D (NAPE-PLD), whereas synthesis of 2-AG occurs through the action of phospholipase C (PLC) and the diacylglycerol lipases alpha and beta (DAGLα and DAGLβ) on membrane phospholipids , phytocannabinoids, and synthetic cannabinoids. Two of the best characterized endocannabinoids are holipids . Once foholipids . Anandamholipids . Endocanholipids . It shouholipids .in vitro and that anandamide was produced by cultured osteoblasts but not osteoclasts. A recent study by the same group reported that the differentiation of human osteoclasts from monocytes is associated with a reduction in 2-AG levels and an increase in anandamide levels  which is an agonist at CB1 and CB2 receptors. It should be noted, however, that many phytocannabinoids such as cannabidiol bind weakly to cannabinoid receptors , p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), PI3/Akt and focal adhesion kinase (FAK) phosphorylation  rather than the Gi/o proteins. The signaling pathways downstream of GPR55 have been less widely studied than CB1 and CB2 but ligand-induced activation of GPR55 has been shown to activate the small GTP binding proteins RhoA, Cdc42, and Rac1  prevents ovariectomy induce bone loss in wild type mice and that CB1 deficient mice are resistant to ovariectomy-induced bone loss, findings which support the view that activation of the CB1 receptor promotes osteoclast differentiation and bone resorption  stimulated human osteoclast formation. The stimulatory effects of AM630 on osteoclast formation in human cultures reported by Rossi and colleagues are exactly opposite to the inhibitory effects if AM630 on osteoclast formation in mouse cultures reported by The CB–1000 nM , whereashibitory , 2008. Fin vitro . Schuehlivatives . In vivoL-α-LPI and O-1602 both inhibited osteoclast formation from bone marrow macrophages in vitro, whereas the GPR55 antagonist cannabidiol increased osteoclast formation. Although GPR55 agonists were found in this study to inhibit osteoclast formation, they actually stimulated the resorptive activity of osteoclasts. Conversely, the GPR55 antagonist cannabidiol enhanced osteoclast formation and inhibited resorptive activity. In keeping with these observations male mice with targeted inactivation of GPR55 were found to have increased numbers of osteoclasts in vivo, but these appeared unable to resorb bone effectively since trabecular bone mass was increased and cartilage remnants at the growth plate were not resorbed efficiently. Rather surprisingly, female mice deficient in GPR55 were found to have reduced numbers of osteoclasts and increased amounts of unresorbed growth plate cartilage, but had normal bone mass  which is structurally similar to both CBD and THC has been found to inhibit formation of multinucleated TRAP positive cells in RANKL treated RAW264.7 cultures and in M-CSF and RANKL treated bone marrow macrophages . This was investigated by 1 deficient mice, but was present in wild type and CB2 deficient mice. In this study, 1 receptors present on presynaptic nerve endings in bone might enhance bone formation by suppressing catecholamine release  CB1 deficient mice on a CD1 background, again confirming that CB1 appears to play a key role in regulating bone formation. Recent findings have shown that the CB1 receptor plays a role in glucocorticoid-induced bone loss  and outbred (CD1) backgrounds exhibited high peak bone mass affecting the trabecular compartment of bone  or peripheral mechanism. This is clinically relevant since if the peripheral effects were predominant, it may be possible to develop agonists of CB1 that do not cross the blood brain barrier that could favorably influence bone formation without causing adverse psychotropic effects. The outcome of these studies will greatly enhance our understanding of the role of the skeletal endocannabinoid system in bone remodeling and encourage the development of new treatments for bone diseases based on targeting cannabinoid receptors.There is a steadily growing body of evidence suggesting that the skeletal endocannabinoid system plays a significant role in regulating bone mass and bone turnover. Several outstanding questions remain unanswered however. One is to define the mechanisms by which endocannabinoid production in bone is regulated and specifically to determine if it is influenced by classical calcium regulating hormones, cytokines and mechanical loading. Further research is also required to fully define the signaling pathways used by these receptors to regulate bone cell activity. There is evidence to suggest that the CBignaling . A furthThe authors are co-inventors on patents claiming the use of cannabinoid receptor ligands as treatments for bone disease.


In early-phase studies with targeted therapeutics and radiotherapy, it may be difficult to decide whether an adverse event should be considered a dose-limiting toxicity (DLT) of the investigational systemic agent, as acute normal tissue toxicity is frequently encountered with radiation alone. We have reanalyzed the toxicity data from a recently conducted phase 1 study on vorinostat, a histone deacetylase inhibitor, in combination with pelvic palliative radiotherapy, with emphasis on the dose distribution within the irradiated bowel volume to the development of DLT.Of 14 eligible patients, three individuals experienced Common Terminology Criteria of Adverse Events grade 3 gastrointestinal and related toxicities, representing a toxicity profile vorinostat has in common with radiotherapy to pelvic target volumes. For each study patient, the relative volumes of small bowel receiving radiation doses between 6 Gy and 30 Gy at 6-Gy intervals (V6-V30) were determined from the treatment-planning computed tomography scans. The single patient that experienced a DLT at the second highest dose level of vorinostat, which was determined as the maximum-tolerated dose, had V6-V30 dose-volume estimates that were considerably higher than any other study patient. This patient may have experienced an adverse radiation dose-volume effect rather than a toxic effect of the investigational drug.When reporting early-phase trial results on the tolerability of a systemic targeted therapeutic used as potential radiosensitizing agent, radiation dose-volume effects should be quantified to enable full interpretation of the study toxicity profile.NCT00455351ClinicalTrials.gov:  With current advances in molecular radiobiology, strategies for improving efficacy of clinical radiotherapy are increasingly focused on investigating targeted compounds as radiosensitizing agents. The accepted investigational sequence for clinical evaluation consists of initial toxicity assessment of the systemic compound in combination with radiation, and the conventional 3+3 expansion cohort design remains the prevailing method for conducting phase 1 trials in cancer therapy [Furthermore, in radiotherapy, toxic complications are both common and acceptable, and adverse events are often interrelated. Radiation-induced early toxicity is commonly experienced as a transient phenomenon either during the therapy course or within a few weeks of treatment completion, typically in normal tissues with a hierarchical proliferative structure, such as the mucosal lining of the gastrointestinal tract . When coIt is widely recognized that irradiation of large volumes is associated with a heightened risk of normal tissue toxicity. For example, in protocols applying irradiation of the bowel with two- to four-field techniques, moderate to severe acute gastrointestinal toxicity, primarily diarrhea, is observed in a significant fraction of patients. Furthermore, the probability and severity of such effects increase with the size of the therapeutic target volume and the dose per fraction . RecentlConsequently, studies that are designed as early investigations into the safety of combining targeted therapeutics with pelvic radiotherapy may be particularly challenging to conduct as acute bowel toxicity is frequently encountered with radiation alone. In this setting, it may be difficult to decide whether or not a toxic event occurring during treatment is greater than might be expected for either of the therapeutic components and specifically whether the event should be considered a dose-limiting toxicity (DLT) of the systemic agent.We have recently conducted a phase 1 study, PRAVO - Pelvic Radiation and Vorinostat, on vorinostat , a histone deacetylase inhibitor, in combination with pelvic palliative radiotherapy for advanced gastrointestinal carcinoma , and havThe PRAVO study was approved by the Regional Committee for Medical and Health Research Ethics and was performed in accordance with the Declaration of Helsinki. Written informed consent was required for participation.The study objective was to determine tolerability of vorinostat, defined by DLT and MTD, when administered concomitantly with palliative radiation to pelvic target volumes. The principal eligibility criterion was pelvic carcinoma scheduled to receive palliative radiation to 30 Gy in 3-Gy daily fractions. Other details on eligibility are given in the initial report . The radToxicity was recorded continuously during treatment and was reexamined two and six weeks after treatment completion, and was graded according to Common Terminology Criteria for Adverse Events version 3.0. DLT was defined as grade ≥3 toxicity. A treatment delay longer than one week due to toxicity was also considered a DLT.Fourteen of the 16 study patients had treatment-planning CT scans visualizing the entire abdominal and pelvic cavities and were evaluable for this reanalysis. Individual loops of small bowel were contoured on each slice of the planning CT scans, enabling the generation of total small bowel dose-volume histograms . For eacThe data reported here is solely descriptive, and no statistical adaptation has been undertaken.Patient baseline characteristics and the complete data on adverse events have been described previously . Of noteSix of the 14 patients experienced grade 3 adverse events; however, in three patients, the reported event was considered to be unrelated to the study treatment: one in a patient receiving 200 mg vorinostat and reporting a grade 3 acneiform rash following commencement of cetuximab, and two in patients at 300 mg vorinostat with pneumonia, who reported grade 3 fatigue that rapidly resolved on antibiotic treatment.The remaining three cases of grade 3 adverse events were considered to be treatment-related and were therefore documented as true DLTs. One of six patients receiving 300 mg vorinostat reported grade 3 anorexia and fatigue. At 400 mg vorinostat, two of six patients reported grade 3 diarrhea, with one patient developing synchronous grade 3 anorexia and hyponatremia and the other experiencing grade 3 fatigue and hypokalemia. Since one of six patients at 300 mg vorinostat and two of six patients within the 400 mg dose cohort reported a DLT, the MTD of vorinostat, according to conventional phase 1 study design, was determined to be 300 mg once daily.For each of the 14 patients, data on absolute volumes of gross tumor, internal radiation target, and total small bowel, relative volumes of small bowel receiving radiation doses between 6 Gy and 30 Gy at 6-Gy intervals (V6-V30), and the daily vorinostat dose is summarized in Table In the vorinostat 400 mg dose cohort, however, the radiation dose-volume records for the two patients reporting DLTs ranked towards the middle of the tabulated list. In these two patients, the relative volumes of irradiated small bowel across all radiation doses (V6-V30) appeared to be within the same order of magnitude and ranked first and third within the vorinostat 400 mg dose cohort separately. Three of the remaining four patients in this dose cohort had considerably lower values of V6-V30.Because some patients had radiotherapy target lesions located in anatomic structures outside the pelvic cavity, such as the perineum or pelvic wall, their irradiated small bowel volumes were smaller than the internal radiation target volumes.As typically may be the case with phase 1 studies, the size of the PRAVO study population was small and few adverse events were recorded. Thus, the resulting data is descriptive and not subject for expedient handling statistically. Nevertheless, following this reanalysis of the PRAVO toxicity data, it seems probable that the single patient reporting a DLT at the vorinostat 300 mg dose level may have experienced an adverse radiation dose-volume effect rather than a toxic effect of the investigational drug. In the remaining four patients within the 300 mg dose cohort, and in all other study patients reported here, the relative volumes of small bowel receiving radiation doses of 12-30 Gy (V12-V30) were substantially smaller. However, our previous conclusion that vorinostat 300 mg once daily defines the MTD in this therapeutic setting  holds trWhen applying the standard 3+3 expansion cohort design to assess relevant normal tissue toxicities in radiotherapy trials, we propose that the potential disease site being irradiated should be clearly specified as study eligibility criterion. Unlike early-phase studies with systemic therapies, where location of disease manifestations presumably is less critical for evaluation of treatment tolerability, the anatomic site of the target lesions determines the normal organs exposed in radiotherapy.The PRAVO study was designed as an initial investigation examining the safety of a histone deacetylase inhibitor employed as radiosensitizing component of pelvic radiotherapy. Importantly, within the study design, small bowel toxicity was an anticipated outcome parameter, since single-agent vorinostat is known to be tolerated at 400 mg daily for continuous dosing, with the most common side effects being fatigue and gastrointestinal toxicities . ConsequThe pragmatic 3+3 expansion cohort design has been the prevailing method of documenting adverse events associated with administration of new drugs, as it requires no modeling of the dose-toxicity curve beyond the classical assumption for cytotoxic agents, including radiotherapy, that toxicity increases with dose . In the Learning from this reanalysis of the PRAVO study outcome data, albeit derived from few reported adverse events in a small study population, radiation dose-volume effects should be quantified when reporting early-phase trial results on the tolerability of a systemic targeted therapeutic used as potential radiosensitizing agent. We believe there are methodological requirements in future early-phase trials utilizing novel radiosensitizers, particularly with regard to patient eligibility criteria, predetermining specific tumor sites and, as a consequence, the radiotherapy target volume.The authors declare that they have no competing interests.ÅB contributed to the design of the study and generated the dose-volume data. SD managed the provision of the clinical information. DH contributed to the concept and design of the study. KF managed the patient database. AHR contributed to the concept and design of the study, managed the patient database, and drafted the final manuscript. All authors contributed to data analysis and interpretation, and read and approved the final version of the manuscript.


Secondary myelodysplasia (MDS) and acute myeloid leukemia (AML) are frequent long term complications in Chronic Lymphocytic Leukemia (CLL) and Waldenström Macroglobulinemia (WM) patients. Although disease-related immune-suppression plays a crucial role in leukemogenesis there is great concern that therapy may further increase the risk of developing these devastating complications.Nucleoside analogs (NA) and alkylating agents are considered appropriate agents in the treatment of both CLL and WM patients. Prolonged immunosuppression related to NA therapy and the incorporation of these agents or their metabolites into DNA, with potentially mutagenic action, leads to speculation that their therapeutic use might be responsible for an increased incidence of second cancer especially when combined with other DNA damaging agents like alkylating agents.In this review the published studies considering the occurrence of secondary MDS and AML in CLL and WM patients are reported and the potential role of chemotherapeutic agents in leukemogenesis is discussed. Advances in the treatment of Chronic Lymphocytic Leukemia (CLL) and Waldenström Macroglobulinemia (WM) have resulted in higher response rates and more durable remissions. Chemoimmunotherapy has become the standard of care and the addition of rituximab to combination chemotherapy is associated with improved outcomes.Some authors have reported an increasing incidence of these late complications over the yearsA comprehensive review of reported myelodysplasia and acute myeloid leukemia (MDS/AML) rates concluded that up to 10% of patients treated for indolent non-Hodgkin’s lymphoma (NHL) using either conventional dose alkylating agents-based regimens or high-dose therapy develop MDS or AML within a decade of primary therapy.In this review we have summarized the possible mechanisms of leukemogenesis, analyzed studies conducted to delineate the incidence of secondary MDS/AML in patients with CLL and WM and discussed the potential relationship between these events and treatment with NA and alkylating agents.Important discoveries have been made over recent years that have contributed to a better understanding of the pathogenesis of neoplasm and in particular of secondary AML/MDS. There are several lines of evidence showing that different mechanisms cooperate in leukemogenesis defining a multistep process.Disease related immunodeficiency, resulting from both cell mediated and humoral-mediated immunity defects, has been postulated as a possible mechanism predisposing to second cancers in non Hodgkin lymphoma (NHL) and in particular in CLL and WM patients. B-cell impairment, low gammaglobulin levels and quantitative and functional defects of various subsets of T cells have all been documented in lymphoprolipherative diseases and are implicated in defective immune surveillance favouring second cancers. In line with this hypothesis is the documented incremented occurrence of malignant tumours in patients having a background of humoral immunodeficiency or abnormalities in T-cell function such as common variable immunodeficiency, ataxia-telangectasia, HIV disease.11However, several studies have raised concerns about the role of therapy in the development of second malignancies and t-MN are a well recognized distinct entity in the 2008 WHO classification of tumors of hematopoietic and lymphoid tissues.12Cytotoxic agents associated with this complication include alkylating agents, topoisomerase II inhibitors, ionizing radiation, antimetabolites and antitubulin agents.Alkylating agents damage DNA either by methylation or DNA inter-strand crosslink formation. The latency between treatment and t-MN is generally long, between 5 and 7 years and may be preceded by a myelodysplastic phase. Most cases present deletion or loss of the long arm or total loss of chromosome 5 (5q−/−5) and/or deletion or loss of the long arm or total loss of chromosome 7 (7q−/−7).16Topoisomerase II is an essential enzyme in eukaryotic cells and is thought to play an important role in DNA replication, transcription and cell division. This enzyme catalyzes the breaking and rejoining of both DNA strands. Topoisomerase II inhibitors block the enzymatic reaction through relegation and enzyme release, leaving the DNA with a permanent strand break. Multiple DNA-strand breaks lead to cell death. Both the antineoplastic effect and the leukemogenic effect are due to chromosomal break-age and this breakage, resolved by chromosomal translocation, is the cause of the leukaemic transformation. A shorter latency of approximately 2 years characterized t-MN secondary to topoisomerase II inhibitors. Balanced chromosome translocations frequently reported after exposure to this chemotherapeutic agents involve 11q23 (MLL) or 21q22 (RUNX1). However, others may also occur, for example t or t15;17) in the case of therapy-related acute promyelocytic leukemia (t-APL). in the c18Antimetabolites share structural similarities with nucleotides, and can be incorporated into DNA or RNA, causing inhibition of cell proliferation. Among antimetabolite agents fludarabine is the nucleoside analogs (NA) more frequently used in the treatment of indolent lymphoproliferative disorders.24In addition to typical genetic abnormalities, t-MN are characterized by molecular changes which may cooperate in the pathogenesis of the disease. FLT3 and p53 mutations are the most frequent described. Chimeric rearrangement of transcription factors, including AML1, CBFB, MLL or RARA and NPM1 mutations have also been reported.25An important role in the multistep secondary leukemogenesis has been recently attributed to the epigenetic changes. Epigenetic mechanisms such as DNA methylation, post-translational modifications of histone proteins and remodelling of nucleosomes affect chromatin structure and contribute to define heritable changes in gene expression. The abnormalities in DNA methylation profile have been described more commonly in therapy-related rather than de novo MDS/AML.26Since only a small fraction of patients exposed to cytotoxic therapy developed t-MN, one of the important questions in the field is what predisposes individuals to develop the disease. A large number of studies have offered evidence demonstrating the importance of genetic variation in key genes, which include those involved in drug metabolism, protection of cellular entities from damage and repair of DNA damage. Mutations in these vital genes are rare while much more common are genetic polymorphisms in detoxification genes and in genes belonging to the major DNA repair pathways.18Although the multistep leukemogenesis mechanism has not been completely clarified, the important knowledge achieved in the last years in this matter has laid the groundwork to a risk adapted therapeutic approach. An adequate screening of individual susceptibility to develop t-MN could influence initial treatment strategies with the goal of decreasing the incidence of these serious complications.WM patients have a well documented increased risk of second cancers and in particular of secondary MDS/AML. Although disease-related immune-suppression has been suggested as one of the main causes of this increasing risk,Several studies have been conducted in Europe and in the United States to retrospectively examine the incidence of secondary MDS/AML in treated WM patients.Alkylating agents and NA such as fludarabine and 2-chlorodeoxyadenosine (2-CdA) have been considered appropriate first line agents for the treatment of WM.28Among patients receiving alkylating agents the incidence of such events ranged from 0 to 6%  in diffeMore recently attention has been focused on long term toxicity of NA-based therapy37Leblond et alIn the first trialThe crude incidence of secondary MDS/AML varied from 1.4 to 8.9% in patients treated with fludarabine alone and was 6% in patients treated with fludarabine combined with cyclophosphamide. Incidence of such events were not different in the two groups of patients. More recently Leleu et alData reported in all these retrospective studies  lead to In the last decade the use of rituximab has also been extensively evaluated in patients with WM and high activity of fludarabine and rituximab combination with or without cyclophosphamide has been demonstrated.Treon et alMore recently toxicities data of fludarabine, cyclophosphamide and rituximab combination (FCR) have been reported by Leblond et alAlthough data on fludarabine treatment are more consistent, recent analysis of long term toxicity after 2-CdA therapy has also been published.The MD Anderson Cancer Center performed a retrospective analysis of 111 consecutive, previously untreated patients with symptomatic WM who received 2 consecutive 4–6-week courses of either 2-CdA alone or in combination with other agents including prednisone, cyclophosphamide, and rituximab. Thirteen patients (12%) developed second malignancies, among them one patient had AML (0.9%). The median time to development of a second malignancy was 85.5 months.43Laszlo et alSecond malignancies are frequent complications in patients with CLL. The most frequent event is CLL transformation to large B-cell non-Hodgkin’s lymphoma also known as Richter’s syndrome.49An increased incidence of these late complications has occasionally been reported in patients receiving alkylating agents. However, occurrence of therapy-related MDS/AML after exposure to alkylating agents seems to be lower when they are used intermittently as opposed to prolonged, continuous exposure.50The study of French Cooperative Group on CLL reported data of two randomized trials in 1535 patients with previously untreated stage A CLL.No evidence of an increased risk of secondary MDS/AML was reported at M.D Anderson Cancer Center in a retrospective analysis of 1374 consecutive CLL patients, even though nearly three quarters of these patients had received prior treatment with alkylating agents.In contrast to the published experience with alkylating agents, secondary MDS/AML are rarely reported following NA as monotherapy in CLL patients.Morrison et al. presented data from intergroup CLL treatment trial comparing treatment with chlorambucil, fludarabine or chlorambucil plus fludarabine, in which a possible increase in the number of cases of therapy-related AML was observed.These data have been recently confirmed by the prospective randomized phase 3 trial, E2997, comparing FC with F alone as initial therapy in 278 CLL patients. With a median follow-up of 6.4 years, there have been 13 cases of t-MN, 9 cases after FC and 4 after fludarabine alone. The increased incidence of t-MN after FC, usually in the absence of additional treatment, suggests that FC is more leukemogenic than fludarabine.58In contrast Robak et al.In the last five years chemoimmunotherapy regimens including rituximab showed high activity in CLL patients improving quality and duration of responses. The chemoimmunotherapy FCR has become a standard treatment for CLL based on the German CLL Study Group (GCLLSG) frontline CLL8 trial and the International REACH trial for patients in first relapse.2Data of long term follow-up of FCR-therapy has been published by Badoux et alMore recently results published by Cancer and Leukemia Group StudyStudies examining consolidative autologous stem cell transplantation in CLL have also reported an increased risk of t-MN. Gribben et al identified in the Dana Farber series, an actuarial incidence of MDS/AML of 12% at 8 years.A lower incidence of secondary MDS/AML was reported by the German CLL-3 trial with three cases in 150 patients who underwent autograft.62As well as the Dana Farber group a high occurrence of MDS/AML has also been described by Milligan et al in patients enrolled in the Medical Research Council Chronic Lymphocytic Leukaemia-5 trial.Recently results published of a phase 3 randomized trial of autografting in CLL versus observation for responding patients after first or second line treatment reported MDS in 4 patients: 3 in the autografiting arm (2.7%) and 1 in the observation arm (0.9%). The relatively low occurrence of MDS in the autotransplantation arm may be related to the limited follow-up of 5 years after randomization.64The increased risk of secondary malignancies occurring in WM and CLL patients is well documented.Overall published data reported frequently higher occurrence of t-MN in WM rather than in CLL patients, while in CLL patients transformation to Richter’s syndrome or the evolution to prolymphocytic leukemia is the more frequent long term complication.The complex immunodeficiency resulting from B-cell impairment, hypogammaglobulinemia, T-cell dysfunction and decreased natural killer-cell activity, associated with these diseases may play a crucial role in leukemogenesis. However, there is great concern that therapy, especially with alkylating agents and purine nucleoside analogs, may further contribute to increase the risk of this devastating complication.Although in the last few years several retrospective studies have been published on this topic, the real impact on risk of secondary MDS/AML complication of the single therapeutic agents has not been definitively clarified.Indeed assessment of the specific risk is often confounded in patients with indolent lymphoprolipherative diseases by frequent exposure to multiple lines of cytotoxic therapy. Rates of secondary myeloid malignancies also vary with the length of follow-up and estimation may be falsely high if early reversible dysplastic features and cytopenia associated with cytotoxic treatment are misinterpreted.Furthermore, the role played by additional immunosuppression due to the introduction of monoclonal antibodies in clinical practice needs to be better understood.These considerations lead to conclude that the impact of secondary MDS/AML and the role of old and new drugs in the development of these late complications needs to be better evaluated in larger prospective studies, especially in young patients. In view of these data, we suggest that risk versus benefit should be considered when treating younger WM and CLL patients.


The mammalian neocortex is composed of a variety of cell types organized in a highly interconnected circuit. GABAergic neurons account for only about 20% of cortical neurons. However, they show widespread connectivity and a high degree of diversity in morphology, location, electrophysiological properties and gene expression. In addition, distinct populations of inhibitory neurons have different sensory response properties, capacities for plasticity and sensitivities to changes in sensory experience. In this review we summarize experimental evidence regarding the properties of GABAergic neurons in primary sensory cortex. We will discuss how distinct GABAergic neurons and different forms of GABAergic inhibitory plasticity may contribute to shaping sensory cortical circuit activity and function. The many varieties of synaptic plasticity provide a high degree of flexibility to neural circuits, allowing organisms to adapt to changing environments and learn from their experiences. In primary sensory cortex different forms of synaptic plasticity can be induced by manipulations of sensory drive, some of which are limited to specific developmental windows , the peptide hormone somatostatin (SOM), or the ionotropic serotonin receptor 5HT3aR  patterns when depolarized and can be classified morphologically as either basket cells or chandelier cells  action potential widths when depolarized, and vertical axons that preferentially target superficial dendritic tufts , distinct biophysical properties . This type of manipulation is typically initiated shortly after birth. Deprivation of one sensory modality can alter cortical circuit activity and responsiveness to sensory stimuli even in sensory areas that are not directly driven by the sensory organ that has been manipulated  mice, GABAergic neurons are only weakly orientation selective  of anesthetized cats, putative FS inhibitory neurons have short response latencies and broad spectral tuning  in rats  and awake rabbits, putative FS inhibitory neurons show broader sensitivity to the direction of whisker deflection and/or to the number of whiskers stimulated than RS units Simons, . In addiFew studies have examined the tuning of GABAergic inhibitory interneurons in primary chemosensory cortices. In the primary gustatory cortex (GC) of anesthetized rats, putative FS inhibitory neurons are primarily tuned to one or two (of four) tastants, almost always including NaCl; while RS units are more broadly tuned and may respond several tastants (Yokota et al., Finally, recent studies have identified putative inhibitory interneuron involvement in multisensory integration and learning. Inhibitory neurons in layer 2/3 of a mouse visuotactile area between visual and somatosensory cortices exhibit less multisensory enhancement of evoked responses than pyramidal neurons, and their activation suppresses multisensory enhancement of activity in pyramidal neurons (Olcese et al., While it is clear that there are lamina and species specific interneuron response properties, some common features exist within cell types Table . FS and In vivo pharmacological manipulation of GABAergic transmission alters receptive fields properties in a number of different species and cortical regions, suggesting a role for cortical inhibition in controlling the stimulus preference and receptive field structure of excitatory neurons (Sillito, The varied response properties of GABAergic neurons make them ideal candidates to modulate the activity of excitatory neurons during sensory processing. Sillito, . HoweverSillito, . DependiThere is general agreement that an inhibitory mechanism contributes to pyramidal neurons having narrower spiking receptive fields than subthreshold receptive fields (Priebe and Ferster, in vivo, coupled with a spike threshold determined in part by inhibitory inputs (Priebe and Ferster, In V1, lateral inhibition could account for many receptive field properties, including orientation tuning, spatial segregation of light and dark receptive subfields, direction selectivity, contrast invariance and suppression. However, it has also been proposed that most of these response properties can be accounted for by a model based on the properties of feedforward, excitatory thalamic inputs that have been observed Inhibitory and excitatory light and dark receptive subfields are largely spatially segregated in the visual cortex of cats Ferster, . The spaOrientation tuning of excitatory and inhibitory inputs to pyramidal neurons has been observed to be either similarly tuned (Anderson et al., Experiments examining the role of inhibition in sharpening tuning width in sensory areas other than V1 have yielded similarly contrasting results. In rodent A1, frequency tuning of inhibitory inputs has been found to be approximately similar to (Wehr and Zador, in vivo may be accounted for by the existence of multiple network architectures existing simultaneously (de la Rocha et al., Experimental evidence in favor of a role for lateral inhibition in sharpening the selectivity of tuning curves has been found, but it has not been observed consistently either within species or cortical regions Table . PossiblThe relative timing of excitatory and inhibitory inputs to a neuron can influence its ability to integrate inputs. Inhibition that follows excitation with a brief delay can create a narrow temporal window within which spiking can occur (Wehr and Zador, In addition to possibly mediating spike timing and tuning curves, inhibition may play a role in response suppression and in maintaining sparseness of spiking responses. Response suppression occurs when a stimulus that would not by itself alter firing of a neuron inhibits the response of that neuron to another stimulus. While GABAergic neurons mediate some forms of response suppression, they likely do not mediate all response suppression. For example, when two tones of the same frequency are played in rapid succession, the cortical response to the second tone is suppressed. As both the onset and offset of stimuli can evoke neuronal responses with transient inhibitory and excitatory components, one hypothesis explaining this effect involves the first tone's offset evoking inhibition that suppresses the response to the second tone (Borg-Graham et al., In V1, a stimulus placed adjacent to a neuron's spiking receptive field can alter its responses to stimuli within the spiking receptive field, a phenomenon referred to as surround suppression. Surround suppression has been shown to be mediated, at least in part, through intracortical inhibition (Ozeki et al., B receptor activation (Pérez-Garci et al., In layer 5 of S1, stimulation of the ipsilateral paw just prior to stimulating the contralateral paw suppresses the neuronal response to the contralateral paw. This suppression is mediated by layer 1 interneurons driven by colossal projections that provide dendritic inhibition via GABAInhibition is ideally suited to contribute to the maintenance of response sparseness. Interestingly, while S1/barrel cortex layer 2/3 pyramidal neurons uniformly show membrane potential responses to whisker stimulation, only ~10% spike during active touch (Crochet et al., in vivo (Murayama et al., Additional circuit precision can be gained through subcellular compartment specific inhibition. While S1 layer 5 pyramidal neurons are rarely connected to each other directly, they are more commonly coupled disynaptically through layer 5 Martinotti cells (Silberberg and Markram, in vivo by optogenetic stimulation of PV positive neurons, suggesting that FS neurons may drive stimulus-evoked activity in the gamma band (Cardin et al., GABAergic neurons do not only contact excitatory neurons, but form networks of electrically and synaptically coupled interneurons, often comprised of the same cell type, that can be activated synchronously (Hestrin and Galarreta, Future work will be necessary to unravel how specific interneuron subtypes contribute to modulating sensory responses, fully understand the roles they play in higher order processing, and address whether there are specific conditions under which lateral inhibition sharpens receptive fields. One recent study in auditory cortex has begun to examine the cell type specific contributions of inhibition to modulating pyramidal cell activity during learning: VIP neurons were found to respond preferentially to reinforcement (Pi et al., GABAergic neurons and synapses undergo a process of postnatal maturation which extends for quite some time after birth and co-occurs with the development of many functional properties of excitatory neurons (Espinosa and Stryker, A receptors and a progressive decrease in a3 and a5 containing GABAA receptors. Significant changes also occur in the expression of γ, δ and ε subunits (Heinen et al., A receptors containing specific α subunits are primarily located in specific subcellular compartments: α1 and α3 containing receptors are enriched at synapses on the soma and perisomatic regions of pyramidal neurons (Klausberger et al., A receptor subunits are similarly localized during development is not known; however, the observed changes in levels of expression suggest that changes in the subcellular targeting of GABAergic synapses may occur.The intrinsic properties of inhibitory neurons change during postnatal development. In rodent V1, PV FS neurons show a progressive decrease in membrane time constant, cell capacitance and action potential width and an increase in intrinsic excitability during the first 2 weeks after eye opening (Goldberg et al., Comparable processes of protracted maturation of inhibitory synapses have been reported in A1 and S1. In both regions there is a progressive increase in spontaneous IPSC amplitude (Kobayashi et al., Developmental changes in inhibitory circuit function go beyond subunit composition, strength and kinetic properties. Inhibitory drive tends to increase during postnatal development (Blue and Parnavelas, The capacity for plasticity at inhibitory synapses in V1 changes sharply during postnatal development at a time point corresponding to the transition between the pre-critical and the critical periods for visual cortical plasticity (Lefort et al., During the developmental window in which inhibitory synapses mature, there are also substantial changes in glutamatergic receptor subunit composition, subcellular localization and function (Carmignoto and Vicini, The maturation of inhibitory synapses is strongly influenced by sensory experience. Visual deprivation induced by raising animals in the dark from birth or shortly thereafter disrupts visual response properties (Benevento et al., Auditory experience also plays a major role in the development of inhibitory inputs in A1, as early hearing loss, either conductive or sensorineural, alters GABAergic neurotransmission (Kotak et al., While whisker plucking prevents the maturation of feedforward inhibition onto excitatory neurons in the barrel cortex (Chittajallu and Isaac, Experiments with early sensory deprivation have shown that sensory experience is necessary for the maturation of inhibitory neurons and their synapses. However, if sensory manipulations are started later in postnatal development, for example during identified sensitive periods whose onset has been shown to correlate with the time course of the maturation of inhibition, sensory cortex is highly sensitive to even brief alterations in sensory drive (Hubel and Wiesel, in vivo during early postnatal development shift the onset of the critical period for visual cortical plasticity in V1. In GAD65 knockout mice, which have low levels of GABA throughout life, the critical period never opens (Fagiolini and Hensch, A receptors containing α1 subunits, which are preferentially located at FS to pyramidal neuron somatic synapses (Fagiolini and Hensch, The maturation of inhibitory synapses has been proposed as a critical step for the modulation of the onset and duration of critical periods (Hensch et al., A receptors (Harauzov et al., Conversely, pharmacologically decreasing inhibition in adult animals can reopen a window for ocular dominance plasticity (Harauzov et al., Alterations in sensory experience are not only dependent upon cortical inhibitory tone to induce plasticity; they also induce specific plastic changes at inhibitory synapses. Altering sensory drive engages inhibitory plasticity differently depending on the age of the animal, the nature of the change in sensory drive, and the specific synapses examined. For example, monocular lid suture induces rapid changes in inhibitory synaptic strength and drive, whether the lid is sutured before or after eye opening (Maffei et al., Removing a single row of whiskers during the first 2–3 postnatal weeks leads to an early potentiation of whisker responses in layer 2/3 of S1 after 3 days of deprivation followed by a loss of responses after 5–10 days (Drew and Feldman, In adult animals one of the main effects of sensory manipulation is the induction of structural changes in inhibitory neurons. A reduced number of dendritic spines has been reported in a subpopulation of inhibitory neurons following adult monocular retinal lesioning (Keck et al., While pharmacologically manipulating inhibition can change the potential for experience-dependent plasticity, global changes in circuit excitability may not be the ideal mechanism for fine-scale refinement of cortical circuit connectivity. Fine scale circuit refinement may require local, possibly synapse specific, regulation of inhibition. The age, layer and cell type specific changes in inhibitory synapses observed following monocular lid suture could provide highly specific modulation of inhibition. The layer 4 FS to pyramidal neuron synapses that are potentiated rapidly following monocular lid suture are sensitive to benzodiazepines, suggesting that they could play a role in critical period regulation (Maffei et al., Experience-dependent reductions in inhibitory drive have been proposed to have a homeostatic role in contributing to the maintenance of neuronal activity in the face of altered sensory drive (Maffei et al., High decibel, high frequency sounds can lead to selective high frequency hearing loss, reorganization of the frequency map in A1 and tinnitus (Yang et al., Whisker removal during a critical period for S1 plasticity reduces feedforward excitatory drive from layer 4 onto both layer 2/3 FS and pyramidal neurons, likely decreasing the activation of layer 2/3 by sensory input (House et al., Different forms of inhibitory synaptic plasticity can be engaged to refine or control local circuits. Despite widespread connectivity (Fino and Yuste, Understanding how changes in GABAergic neurons and their synapses contribute to changing sensory responses and maintaining homeostasis of firing rates in the face of altered sensory drive will be essential to pushing the field of experience-dependent plasticity forward. Recent work has shown that a brief unilateral reduction in visual drive strongly modulates the binocularity of FS inhibitory neurons. While FS inhibitory neurons are normally more binocular than pyramidal neurons Swadlow, , soon afAt first glance, studies showing that monocular lid suture does not affect the ratio of excitatory and inhibitory inputs to layer 4 pyramidal neurons (Iurilli et al., in vivo favors the induction of plasticity is not known. Like lid suture, long term monocular strabismus disrupts ocular dominance (Hubel and Wiesel, Two particularly exciting studies have shed light on possible roles of inhibitory plasticity in modulating sensory responses. In addition to reducing inhibition in layer 2/3, whisker trimming significantly reduces gamma oscillations in the circuit driven by the deprived whisker (Shao et al., Changes in inhibitory drive are certainly not the sole mechanism at play in the experience-dependent rewiring of sensory cortex, as trophic factors, excitatory synapses, neuron intrinsic properties and extracellular matrix interactions have all been shown to contribute to the altered visual response properties and ocular dominance (Berardi et al., The maturation and plasticity of GABAergic neurons play fundamental roles in the development of sensory cortex. Inhibitory synaptic transmission and plasticity may affect critical period timing, specific receptive field properties, spike timing and other temporal aspects of coding, gain, neural synchrony, spontaneous and evoked firing rates, the capacity for plasticity at other synapses, and contribute to the homeostasis of circuit excitability. The role of all the distinct population of inhibitory neurons in the experience-dependent reorganization of cortical circuits has not been fully explored, but there is sufficient evidence to show that different inhibitory neuron types may be engaged to modulate different aspects of neuron response properties. The challenge ahead lies in identifying the distinct roles of inhibitory neurons in cortical circuits and in determining how their plasticity contributes to modulating other neurons' response properties, connectivity, and capacity for plasticity. Development of new tools and experimental approaches to selectively manipulate specific mechanisms of inhibitory plasticity will be instrumental for determining their role in sensory cortex development and function.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.


Carbon nanotubes (CNTs) are considered as promising materials for nanomedicine applications in general and have been recently tested toward excitable cell growth. CNTs are cylindrically shaped structures made up of rolled-up graphene sheets, with unique electrical, thermal, and mechanical properties, able to effectively conducting electrical current in electrochemical interfaces. CNTs-based scaffolds have been recently found to support the  The development of innovative materials able to effectively interface biological tissues and to tune and boost their functional performance is an active and heterogeneous field of current research, and nanotechnological approaches for interfacing excitable cells are disclosing very promising perspectives Silva, . In partCNTs are emerging as an ideal material to be interfaced with electrically active tissues as neuronal and cardiac tissues. This relatively recently discovered class of materials possess high electrical and thermal conductivity, strong mechanical resistance and elasticity and have demonstrated good biocompatibility in many biological studies. Moreover it has been shown that the functionalization of CNT walls can tune their biological properties and their metabolic fate  and skeletal-Actin (Sk-Actin). Genes associated to a more mature phenotype like α MHC are significantly increased in cardiomyocytes grown on CNTs compared to gelatin controls after 3 days culturing, while at the same time, the levels of pathological cardiac hypertrophy markers (β MHC and Sk-Actin) were unaffected. Therefore, after an initial pro-proliferative profile, CNTs scaffolds promote a “mature gene expression profile” of cardiomyocytes over time  cells survival and growth has been investigated. In 2006, Garibaldi and colleagues exposed cardiac muscle cells from the H9c2 (2-1) heart cell line to pure single-walled nanotubes suspended in culture medium, and observed no short-term toxicity of soluble CNTs  can boost, within a physiological range, cardiac cells maturation, while supporting and promoting cellular viability and adhesion, stimulating a pro-proliferative program and promoting cell differentiation. Furthermore, CNTs scaffolds are able to exert a protective role for cardiomyocytes against pathological stimuli . All these properties make CNTs excellent candidates for the development of innovative, effective, and performing devices to interface and engineer cardiac tissue, with the added value of the intrinsic electric conductivity of the scaffold itself. The promising exploitation of CNTs-based platforms for cardiac applications is further encouraged by the growing amount of data on CNTs biocompatibility and impact on immune responses. Serious concerns have been raised about CNTs safety as toxic effects were reported after CNTs injection in lungs (Poland et al., 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.


FOXQ1, MAST2, and CDH4, were found to play a role in hair follicle development and human cancer, signal transduction, and tumor repression, respectively. These showed strong indications of natural selection in Philippine Negritos, a group of aboriginal hunter-gatherers living in the Philippines. MTTP, which has associations with metabolic syndrome, body mass index, and insulin regulation, showed a strong signature of selection in Southeast Asians, including Indonesians. Functional annotation analysis revealed that genes and genetic variants underlying natural selections were generally enriched in the functional category of alternative splicing. Specifically, many genes showing significant difference with respect to allele frequency between northern and southern Asian populations were found to be associated with human height and growth and various immune pathways. In summary, this study contributes to the overall understanding of human local adaptation in Asia and has identified both known and novel signatures of natural selection in the human genome.Genetic studies of human local adaptation have been facilitated greatly by recent advances in high-throughput genotyping and sequencing technologies. However, few studies have investigated local adaptation in Asian populations on a genome-wide scale and with a high geographic resolution. In this study, taking advantage of the dense population coverage in Southeast Asia, which is the part of the world least studied in term of natural selection, we depicted genome-wide landscapes of local adaptations in 63 Asian populations representing the majority of linguistic and ethnic groups in Asia. Using genome-wide data analysis, we discovered many genes showing signs of local adaptation or natural selection. Notable examples, such as  HBB and G6PD, whose mutants and deficiency alleles were found to confer resistance to malaria respectively, LCT, whose variant allows lactose tolerance to persist throughout adulthood, SLC24A5, which contributes to skin pigmentation diversity, and EDAR, one of whose polymorphisms may cause variation in hair morphology High-throughput DNA genotyping and sequencing technologies and novel statistical approaches advanced genome-wide detection of molecular signature of natural selection in the human genome NAT2 gene supporting acetylation in human adaptation to farming in Central Asia, positive selection on NRG-ERBB4 pathway in Middle East, natural selection on an ABCC11 SNP determining earwax type in East Asians, and EPAS1 and EGLN1, which are likely to be responsible for high-altitude adaptations in Tibetans However, most of the previous genome-wide studies of natural selection have concentrated on population samples collected from international collaborative efforts such as the Human Genome Diversity Panel (HGDP), the International HapMap Project, and the 1000 Genome Project We therefore attempted to provide the first comprehensive landscape of local adaptations in Southeast Asia using genome-wide analysis of 63 populations obtained from the HUGO Pan-Asian SNP consortium, where 54,794 autosomal single-nucleotide polymorphisms (SNPs) in 1513 individuals were genotyped using Affymetrix GeneChip Human Mapping 50K Xba Array Taking advantages of the dense population samples, we drew a general yet comprehensive picture of human local adaptation, particularly in Southeast Asia. First, we characterized an overview of putative local adaptation signals using between-population comparisons, which provided a guide for further, finer-scale examinations. However, since the marker density was not sufficiently high in our data, we mainly focused on allele-frequency-based analysis rather than haplotype-based analysis. We classified populations into several sub-groups according to the genetic structures revealed in a previous study After removing some populations showing evident admixture as reported in a previous study ST, of genome-wide SNP markers in each pair. As the total sample size of each group was not always similar, some groups might have much larger or smaller sample size than others. We randomly sampled the same number of individuals from each group so that the sample size of groups under comparison is comparable. The most commonly used means of identifying putative signals of selection has been the outlier-based method ST values within genes. In order to control the bias resulting from the number of genotyped SNPs or the size of genes, here we employed a sliding window strategy  ST-based approach also appeared on the list produced by the XP-CLR test  was best illustrated by our functional analysis . The OMIPLCG2 with the SNP in the gene reaching FST of 0.55. These findings indicated that signaling pathways have been under natural selection and might have played an important role in Negritos adapting to local environment in Malaysia.Another group of Negritos in Malaysia showed a great variation in keratinocyte differentiation and epidermal cell differentiation compared with Indonesian population (Benjamini FDR corrected p<0.01). There were a series of KEGG signaling pathways showing enrichment of divergence between the Negritos and Indians , for exaIn summary, giving clues to biological basis of regional natural selection, our functional analyses provided a great deal of insight into human local adaptation based on allele frequency differentiation between populations. A similar procedure of functional analysis was also performed upon signals identified by XP-CLR analysis .ST value among that of all the SNP markers within it. We chose the top ten candidates from each group pair, as shown in In order to better understand selective pressures upon Asian populations that might not have previously been evaluated, we ranked candidate genes according to the highest FMLKL, PPP1CC, DAPP1, and ABCA12.Considering the many group comparisons and candidate genes, we only highlighted the top candidate gene from each pair, as shown in FOXQ1 showing the strongest signal of positive selection in comparisons with most other Asian groups was involved in the development and morphogenesis of the hair follicle. In Malaysian Negrito population, the outstanding candidate WNT4 had something to do with gamete generation and specificity, indicating the influence of selection on human reproduction. Additional candidate genes specific to some groups were also identified  on chromosome 20. The whole genomic picture of selective signals in these comparing pairs confirmed the most significant signature of local adaption in Philippine Negritos  and southern populations . MTTP protein catalyzes the transport of lipoproteins in the process of lipid metabolism and its variants are associated with plasma cholesterol levels and body mass index DAPP1 protein, an adaptor protein, regulates antigen receptor signaling downstream of phosphatidylinositol 3-kinase and is closely related to human immune system ST scores upon the whole genome loci in the comparisons between Southeast Asian populations and northern populations  showed remarkable evidence of selection signatures  and southern populations (Indian)  . This inIn this study, we performed the first comprehensive genome-wide scan for natural selection in 63 Asian populations and identified a number of putative selection signals. These may further the understanding of human local adaptation in Asia. One of the great advantages of our data is the fact that it was produced by sampling populations of a high geographic resolution and covering a large number of Asian populations, particularly Southeast Asian populations.To draw a general picture of genome-wide signs of local adaptation in Asian populations, we investigated significant SNPs underlying population differentiation with respect to allele frequency. Using the sliding-window-based approach, we adopted a 1% genome-wide cutoff with respect to population differentiation across windows and indicated top 200 genes as putative selection signals in each group pair. Our results showed that Negritos and Indians had the most significant divergence regarding the allele frequency. Further, functional analysis of candidate genes suggested that natural selection among Asian populations is likely to induce mutations that play roles in alternative splicing. Selection divergence from northern Asian populations and southern Asian populations aimed at genes related to cadherin and a plenty of immune and related disease pathways. Another intriguing enrichment was about biological pathways of human height and growth, making Japanese and Korean populations different from Philippine Negritos. Moreover, Philippine Negritos and Malaysian Negritos demonstrated great genetic divergence and carried genes with polymorphisms whose allele frequency differed from other Asian populations.Despite the fact that we did have observed some enrichment of genes, a part of categories, mainly for cell adhesion-related terms, do harbor many genes from protocadherin gamma gene cluster that are physically closely located in a region of 100 kb . AlthougST approach and better understand the selective signals, we turned to the results of XP-CLR analysis, and highlighted notable examples of candidate genes showing large numbers of extreme SNPs achieving peak FST and XP-CLR scores  and southern populations , which was supported by FST-based analysis. We believe that these genes merit further study because they are all important for human development or immunity, especially PIK3R3, since it is involved in the insulin receptor signaling pathway, which has been suggested as an adaptive pathway among African Pygmies To confirm the reliability of the candidate signals identified by FR scores . GeneralHLA region varies dramatically across broad geographical populations, and it has been found to be a predictor of northern versus southern ancestry in Europe and other parts of the world HLA showed notably high FST and XP-CLR scores in the comparisons of certain Asian populations, especially between Indians and SouthernChinese&Thai1, and between Han Chinese populations and southeastern populations  (IFNG and LEPR reflected the difference between northern and southern Asian populations. As people who reside at higher latitudes (north) are generally taller than those living at lower latitudes (south), these two genes may be in association with the stature of Asian individuals.In this study, our results provided a great deal of evidence for local adaptation in Southeast Asian populations that burden historical and geographical admixture, and in Negritos that have received increasing amounts of attention from the research community. Previous studies have identified some significant and comprehensive local adaptation signatures in African pygmies that are similar to Negritos on stature but differ substantially in genetic makeup. Most of the signatures lie in the immunity-related genes because of the different microbial environments in different habitats. For example, the e&Thai2) . Our fune&Thai2) . EspeciaTYRP1 expression in response to differential UV radiation based on latitude DDB1, which protects the skin from solar UV exposure, has different alleles fixed in continents from different latitudes Considering the differences between northern and southern populations, the most remarkable and comprehensive clue may be related to skin pigmentation. It has been proved, from Africa to eastern Europe and eastern Asia, that microRNA regulation acts as a rheostat to optimize However, this study has two principal limitations. The first is the low SNP density relative to the high-density data available in public resources and therefore may not fully represent the entire genome. But these SNP markers randomly distribute across the genome, thus providing a background of genome-wide information Despite its limitations, our study can be regarded overall as a useful guide for further evaluation of local adaptation in Asian populations. The strongest signs of local adaptation in Asian populations may have been novel candidates, suggesting the advantage of data, such as the data evaluated here, which cover wide geographic areas within Asia. This may be particularly true of candidates discovered among the Negritos. A more comprehensive collection of greater numbers of samples across a wider geographic range will be necessary for further studies of positive selection, especially in the populations whose members exist across special geographic regions, such as the Negritos and Tibetans. More importantly, high-resolution data such as genome-wide SNP data and next generation sequencing data will facilitate investigation of the genetic basis of local adaptation in a more effective and detailed way than has previously been possible. The development of novel and sophisticated methods of deciphering signs of natural selection will assist researchers and improve the biological understanding of human adaptive evolution.The HUGO Pan-Asian SNP consortium constructed a database (PanSNPdb) containing data of human genetic diversity among Asians by sampling 1,719 unrelated individuals among 71 populations from China, India, Indonesia, Japan, Malaysia, the Philippines, Singapore, South Korea, Taiwan, and Thailand ST following Weir and Hill ST value of the top 3 SNPs in each window as the value of it and defined 1% tail of the distribution as the threshold. Those windows above were considered regions underlying local adaptation. After that, for each individual window, we sorted SNPs based on their FST and selected the top n SNPs (n = int(N/i), N represents the total number of SNPs in a certain window. If N<20, i = 5; if N> = 20, i = 10), which were thought to be candidate SNPs. Finally we mapped these candidate SNPs onto genes and picked out the top 200 genes as candidate genes for local adaptation.To detect signs of local adaptation in Asian populations, we used an allele-frequency-based approach. For each pair of groups, we calculated unbiased estimates of FST analysis, we applied another allele-frequency-based method: the cross-population composite likelihood ratio test (XP-CLR) To confirm selection signals identified by Fhttp://david.abcc.ncifcrf.gov/) to perform functional analysis upon candidate genes acquiring significant SNPs in each pair of group comparisons We implemented DAVID Bioinformatics Database Click here for additional data file.Figure S2Signatures of local adaptation in Southeast Asian populations associated with MTTP and DAPP1.(PDF)Click here for additional data file.Table S1Characterization of datasets and group division.(PDF)Click here for additional data file.Table S2Selection candidates detected by XP-CLR approach.(XLSX)Click here for additional data file.Table S3Candidate genes overlapping with XP-CLR genes.(XLS)Click here for additional data file.Table S4Functional enrichment analysis.(XLSX)Click here for additional data file.Table S5Top 10 candidate genes in 36 comparing pairs.(XLS)Click here for additional data file.Table S6ST approach.Candidate genes underlying local adaptation identified by F(XLS)Click here for additional data file.Table S7Outstanding candidate genes demonstrating strong signs of local adaptation in Asia.(XLS)Click here for additional data file.Table S8Candidate genes overlapping with 14 literature genes.(XLS)Click here for additional data file.Text S1Results of XP-CLR analysis.(PDF)Click here for additional data file.


Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, characterized by the progressive loss of motor neurons. The cause of this selective neuronal death is unknown, but transcriptional dysregulation is recently emerging as an important factor. The physical substrate for the regulation of the transcriptional process is chromatin, a complex assembly of histones and DNA. Histones are subject to several post-translational modifications, like acetylation, that are a component of the transcriptional regulation process. Histone acetylation and deacetylation is performed by a group of enzymes (histone acetyltransferases (HATs) and deacetylases, respectively) whose modulation can alter the transcriptional state of many regions of the genome, and thus may be an important target in diseases that share this pathogenic process, as is the case for ALS. This review will discuss the present evidence of transcriptional dysregulation in ALS, the role of histone deacetylases (HDACs) in disease pathogenesis, and the novel pharmacologic strategies that are being comprehensively studied to prevent motor neuron death, with focus on sirtuins (SIRT) and their effectors. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the death of upper and lower motor neurons. There are two type of ALS based on the presence of an identified mutation as a cause of the disease. The familial type (fALS) is most commonly caused by mutations in the superoxide dismutase type 1 (SOD1) gene, for which there is a widely studied transgenic mouse model; however fALS represents only about 10% of cases. The most common type of ALS is called sporadic (sALS), but because there are no identified etiologic factors, validated experimental models lack for this type of the disease. Nevertheless, disease progression and pathologic characteristics in both, fALS and sALS, are similar. Also, in both types of the disease, motor neuron degeneration has been associated with the same mechanisms, such as glutamate-mediated excitotoxicity, inflammatory events, axonal transport deficits, oxidative stress and mitochondrial dysfunction  to the transcriptional machinery, ultimately affecting gene expression , their distribution and the evidence of their role in ALS. The role of SIRT  will be addressed later.2+-dependent enzymes. Class I include HDAC1, HDAC2, HDAC3 and HDAC8, which are localized in the nucleus and ubiquitously expressed in mammalian tissues . Broadly speaking, these enzymes are involved in the regulation of gene-specific transcription through the formation of stable transcriptional complexes  resulting in increased survival , sodium butyrate, trichostatin A, 4-phenyl butyrate, MC1568 –class II selective–, and tubucin –HDAC6 specific–), and benzamide-based . Their potencies and selectivities vary, but most of them can be considered pan-HDACs inhibitors  in the spinal cord  due to its numerous mechanisms of action, including HDAC inhibition , the active form of the inhibitory subunit of NF-κB (i-κB) and of beta cell lymphoma 2 (bcl-2) proteins, all involved in survival and stress responses , another pan-HDAC inhibitor, was shown to extend survival and motor performance in the transgenic ALS SOD1 animal model, and these effects were attributed to an upregulation in the expression of nuclear factor +) to perform their catalytic deacetylation and mono-ADP-ribosyl transferase activities  with various cellular localizations and preferred enzymatic targets, but what makes unique this class is the dependence of nicotine adenine dinucleotide (NADSIRT are named after the silent information regulator 2 (sir2) gene in yeast. When overexpressed, the product of this gene extended the lifespan of budding yeast by repressing genomic instability ; SIRT1 expression is confined to the neuronal nucleus in parvalbumin (GABAergic) and tyrosine hydroxylase (dopaminergic) neurons , particularly resveratrol, which is present in grapes and in red wine, and SRT1720, a synthetic compound derived from resveratrol. These compounds activate selectively both SIRT1 and SIRT2, whereas activators of the remaining SIRT have not yet been described  infusion in the lumbar spinal cord , targeted expression of FoxO3a to the nucleus (where it can modulate the transcription of its target genes) by genetic or pharmacologic means prevented neuron death  in astrocytes was noted in the double transgenic, suggesting that PGC1-α could exert its beneficial effects through other mechanisms (Liang et al., α protected against mitochondrial fragmentation and neuronal cell death, although the authors did not demonstrate the relation between SIRT3 and PGC1-α.Another well studied SIRT1 effector is PGC1-o et al.  used a t+ cellular levels (Liu et al., These findings indicate that sirtuin activation protects against motor neuron degeneration, although some findings in different experimental models suggest that activators may protect through other mechanisms Tang,  or even α. Not all types of HDACs have been evaluated in ALS models and the effects, toxicity, dosage, timing, and mode of administration of specific drugs that inhibit HDAC or activate SIRT have not been established. Of note is the fact that the most widely used model of ALS in which these strategies have been studied is the transgenic rodent with mutations in the human SOD1 gene, but this represent only about 2% of all ALS cases. Novel experimental models of motor neuron degeneration in vivo are clearly needed before safely moving these drugs to clinical trials for a disorder that has proved recalcitrant to all, past and current, therapeutic procedures.The opportunities for a really effective therapy for ALS are scarce, and therefore HDAC inhibition and sirtuin activation merit further investigation. Studies on the modification of the activity of these enzymes in in vitro and in vivo experimental models of neurodegeneration, including ALS, have given valuable information suggesting potential therapeutic opportunities for this disease. Nevertheless, the mechanisms underlying such effects are poorly understood and their clarification is necessary for designing more specific and potent treatment strategies. Accumulated evidence on sirtuin activation has provided valuable information about the molecular pathways that could be relevant to halt motor neuron degeneration, such as those related to cellular energetic state and PGC1-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.


Besides demonstrated efficacy, selective serotonin reuptake inhibitors (SSRIs) hold other advantages over earlier antidepressants such as greater tolerability and a wider range of clinical applications. However, there is a growing body of clinical evidence which suggests that SSRIs could, in some cases, be associated with a withdrawal reaction upon cessation of regular use. In addition to sensory and gastrointestinal-related symptoms, the somatic symptoms of the SSRI discontinuation syndrome include dizziness, lethargy, and sleep disturbances. Psychological symptoms have also been documented, usually developing within 1–7 days following SSRI discontinuation. The characteristics of the discontinuation syndrome have been linked to the half-life of a given SSRI, with a greater number of reports emerging from paroxetine compared to other SSRIs. However, many aspects of the neurobiology of the SSRI discontinuation syndrome  remain unresolved. Following a comprehensive overview of the clinical evidence, we will discuss the underlying pathophysiology of the SSRI discontinuation syndrome and comment on the use of animal models to better understand this condition. Selective serotonin reuptake inhibitors (SSRIs) are widely used in the treatment of depressive disorders . For example, using the Discontinuation Emergent Signs and Symptoms (DESS) checklist, Rosenbaum et al.  assessedn = 56). These symptoms included dizziness (50% of patients), increased dreaming/vivid dreams (35%), fatigue (30%), nausea/vomiting (30%), headache (25%), anxiety/agitation (25%), paresthesia (15%), insomnia (10%), diarrhea (10%), visual disturbances (10%), fever (10%), tremor (5%), irritability (5%), and chills (5%). In addition to these symptoms, in an assessment of 87 patients who had their treatment interrupted for 4–7 days in a double-blind placebo study, Hindmarch et al. . Overall, the crucial question of whether the discontinuation syndrome is equally related to the lack of specificity and/or the neurobiology related to the half-life of the SSRI remains unclear. Potential genetic-drug interactions should also be systematically assessed in future studies. Indeed, a recent clinical study suggested a possible involvement of the C(-1019)G polymorphism of the serotonin 5-HT1A) and nerve terminals (5-HT1B) were blocked. Incidentally, the acute effects of SSRIs on serotonin neurotransmission are controlled by several feedback mechanisms, which include 5-HT1A and 5-HT1B/1D autoreceptors  to achieve therapeutic efficacy. This delay is thought to partly reflect the time required for autoreceptors to desensitize so as to facilitate serotonin neurotransmission , multiple targets  are likely to be involved in the mechanism of action of SSRIs  does not appear to be required for the beneficial anxiolytic/antidepressant-like effects of chronic fluoxetine , followed by a discussion of the potential molecular mechanisms involved. Despite the relatively consistent cluster of clinical symptoms observed in the SSRI discontinuation syndrome, few animal studies have been conducted to explicitly examine the effects of withdrawal from chronic SSRI treatment. Moreover, most of those studies have used fluoxetine, despite a low incidence of discontinuation syndrome associated with this specific SSRI in the clinic.In one animal study, contrasting with the reduction of locomotor activity observed during chronic administration of fluoxetine (30 mg/kg/day for 5 days), fluoxetine-treated rats showed a significant increase in locomotor activity upon discontinuation . These studies suggest that the best route of action for cessation of SSRI treatment is to taper down the dose of the medication rather than abrupt termination, as tapering is likely to decrease the possibility of the occurrence of discontinuation symptoms. Due to the broad range of symptoms which can develop as part of the discontinuation syndrome, prior to drug initiation, patients and caregivers need to be provided with adequate education and realistic, objective appraisals of possible outcomes which can develop during and following antidepressant treatment.The current evidence suggests that the discontinuation syndrome is dependent on the SSRI half-life, with more reports of symptoms occurring in patients treated with paroxetine compared to other SSRIs. However, studies designed to assess the onset of discontinuation syndrome, and how the syndrome coincides temporally with pharmacokinetic withdrawal, are still lacking. Such studies are critical in order to draw conclusions when comparing SSRIs with short versus long half-lives. It is currently unclear as to whether the discontinuation syndrome is equally related to the pharmacological properties of a given SSRI, and/or its half-life. Future studies could also examine possible associations of the syndrome with clinical characteristics, as one study reported that the discontinuation syndrome was more common in patients with earlier onset of dysthymic disorder, and was also more common in females  experience discontinuation symptoms. In that regard, a recent clinical study indicated a possible involvement of the C(-1019)G polymorphism of the serotonin 5-HTThe 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.


N-acetyl-D-mannosamine, a naturally occurring precursor of sialic acid, improves sialylation of Angptl4 in vivo, and reduces proteinuria by over 40%. By contrast, a sialylated circulating form of Angptl4, mostly secreted from skeletal muscle, heart and adipose tissue in all major primary glomerular diseases, reduces proteinuria while also causing hypertriglyceridemia. Intravenous administration of recombinant human Angptl4 mutated to avoid hypertriglyceridemia and cleavage has remarkable efficacy in reducing proteinuria by as much as 65% for 2 weeks after a single low dose. Both interventions are mechanistically relevant, utilize naturally occurring pathways, and represent new generation therapeutic agents for chronic kidney disease related to glomerular disorders.Current drugs used to treat proteinuric disorders of the kidney have been borrowed from other branches of medicine, and are only partially effective. The discovery of a central, mechanistic role played by two different forms of the secreted glycoprotein angiopoietin-like 4 (Angptl4) in human and experimental glomerular disease has opened new treatment avenues. Localized upregulation of a hyposialylated form  of Angptl4 secreted by podocytes induces the cardinal morphological and clinical manifestations of human minimal change disease, and is also being increasingly recognized as a significant contributor toward proteinuria in experimental diabetic nephropathy. Oral treatment with low doses of  Current therapy for kidney disease in general and kidney disease related to proteinuric disorders in specific has relied upon the use of agents borrowed from other fields. One category of agents used to treat glomerular disease have immunosuppressive properties, and include glucocorticoids, cyclophosphamide, azathioprine, chlorambucil, mycophenolate mofetil, cyclosporine, tacrolimus, and the anti-CD20 antibody. Another category contains drugs used for supportive therapy, including a variety of diuretics and agents that block the renin angiotensin system at different levels, like angiotensin converting enzyme inhibitors, angiotensin receptor blockers, spironolactone, and more recently, renin inhibitors like aliskiren. The traditional rationale behind the use of the first category of drugs was their immunosuppressive effect, but it has become clear over the past decade that many of these drugs have direct effects on resident glomerular cells . The conFigure 1). By grouping hypoalbuminemia with proteinuria and lipiduria with hyperlipidemia, we used three functional components of nephrotic syndrome for our studies: proteinuria, hyperlipidemia (hypertriglyceridemia and hypercholesterolemia), and edema. Once a gene involved in at least two of these three components was identified, its molecular pathways were dissected, and therapeutic strategies were developed specifically to reduce proteinuria without aggravating the other components of nephrotic syndrome. During discovery phase experiments : (a) A hyposialylated form secreted from podocytes in MCD  reduces proteinuria , transient upregulation after the onset of proteinuria in experimental membranous nephropathy (MN), and no change in podocyte expression in non-HIV collapsing glomerulopathy (CG) and focal and segmental glomerulosclerosis (FSGS) . Furthers in MCD , and lats in MCD . Conversteinuria , and thiteinuria  and diabteinuria . (b) A nteinuria . Most ofteinuria .Since circulating Angptl4 is a major molecular mediator of nephrotic syndrome due to a variety of glomerular diseases, it is an ideal candidate for a parenteral once-a-month therapeutic agent to treat proteinuric disorders that cause chronic kidney disease. On the other hand, ManNAc is orally bioavailable, and has enormous potential in low doses to treat diabetic nephropathy and as a maintenance drug to prevent MCD relapse after the first episode is treated with glucocorticoids.in vivo. Podocytes in NPHS2-Angptl4 transgenic rats, and PAN rats in the early glucocorticoid-sensitive phase overproduce a hyposialylated form of Angptl4. Treating these rats with ManNAc improves the sialylation of Angptl4 in vivo, and reduces proteinuria by at least 40%. By contrast, 2D gel migration pattern of podocalyxin, a sialylated structural protein expressed in podocytes, is not altered in a manner to suggest an effect on structural proteins . It is important to understand the differences between structural and secreted proteins in terms of their requirement for sialic acid. A substantial amount of sialic acid in cells is recycled , which reduces tremendously the burden for de novo sialic acid synthesis , since there is no major nutritional source of N-acetylneuraminic acid (NANA/Neu5Ac), the predominant form of sialic acid present in humans (N-glycolylneuraminic acid (NGNA/Neu5Gc) using the enzyme NANA hydroxylase, which is non-functional in humans due to an exon deletion/frame shift mutation , thereby surmounting the intrinsic limitations of the pathway during high demand states. Diseases involving the podocyte are especially suited for ManNAc therapy for two reasons. Unlike most other cells in the body, podocytes divide very infrequently, which sets the stage for accumulation in these cells and consequently lower oral dose requirement when compared to therapy targeting dividing cells. Rapid urinary excretion after oral dosing  to link proteinuria with hypertriglyceridemia . AlbuminFigure 4). As proteinuria reaches nephrotic range, hypoalbuminemia develops, and a combination of high FFA containing albumin and hypoalbuminemia raises the plasma ratio of FFA to albumin . The precise mechanism by which the Angptl4 – αvβ5 integrin interaction reduces proteinuria is not known, but it is possible that additional feedback loops within the glomerulus are involved. Second, Angptl4 inactivates LPL activity in these organs, thereby reducing the conversion of triglycerides into FFA, which reduces FFA uptake by this pathway , and also results in hypertriglyceridemia. In summary, the retention of FFA in nephrotic syndrome tends to accelerate production of Angptl4 from peripheral organs, whereas its own effect on LPL activity in these same organs limits the extent of upregulation . Since native recombinant human Angptl4 in circulation will have the same limitations as tissue secreted Angptl4, it is appropriate to modify the protein to ignore the local feedback loop (“the brake”), while acting solely on the systemic feedback loop (“the accelerator”). This would allow for achieving higher circulating Angptl4 levels than otherwise possible by using existing agents, without incurring the risk of hypertriglyceridemia or starving skeletal muscle and heart of energy substrate. Since population based studies . Also, since Angptl4 readily cleaves between amino acids 161 to 164, we made additional changes in this region to improve the half-life of the intact protein. Studies with recombinant rat Angptl4 have shown the formation of very high order oligomers that migrate even slower than α2-macroglobulin (720 kDa) on one-dimensional non-reducing gels , including pharmacokinetic studies in nephrotic rats, show that mutagenesis did not reduce the ability to reduce proteinuria in Buffalo Mna rats (a model of FSGS) and Zucker diabetic fatty rats . Some of the mutant proteins reduced proteinuria more substantially and for a longer duration than the wild type protein . Plasma triglyceride levels increased with the wild type, but not the mutant proteins. A single 55 μg dose of recombinant human Angptl4 reduced proteinuria for over 2 weeks, with peak reduction of up to 65% in Buffalo Mna rats . Finally, a smaller dose (15 μg) of wild type recombinant Angptl4 in ZDF rats reduced proteinuria without causing hypertriglyceridemia , suggesting a lower threshold for Angptl4 to act via the systemic loop, compared to the local feedback loop. These recombinant proteins are now being produced on a large scale to allow for expanded testing and prolonged administration in nephrotic rats to study effects on chronic kidney disease. Since Angptl4 is a normal circulating protein with levels that vary several fold with fasting / feeding, the likelihood of major aberrant side effects of therapy with mutant Angptl4 protein is relatively low. However, like any mutated protein used for therapeutics, there is a potential for developing an antibody response in some patients that may limit long term usage in these patients.Another way to manipulate this naturally occurring pathway is to use recombinant human Angptl4, which would be superior to the agents mentioned above, since both therapeutic and side effects will be selective for this protein  suggests that manipulating Angptl4 related pathways in the context of therapeutics has a high chance of success.In summary, sialylation based and recombinant mutated Angptl4 based therapeutic strategies hold significant promise in the treatment of common forms of proteinuric chronic kidney disease, including diabetic nephropathy. Both areas are novel, protected by intellectual property, innovative, mechanism based, and have been extensively studied The discovery of Angptl4 as a major player in human nephrotic syndrome was based on a strategy to identify and selectively investigate genes/ proteins that could potentially link at least two of the three major components  of nephrotic syndrome. This approach continues to have potential in the future, since the pathogenesis of hypercholesterolemia, proteinuria, and increase peripheral capillary permeability related to edema are not completely understood. Also, the reason for the muted response of the liver to counter hypoalbuminemia in nephrotic syndrome by increasing albumin synthesis in most patients is unclear. In developing future therapies, it is important to steer clear of old unyielding hypotheses, like Shalhoub’s hypothesis of putative T-cell secreted factors as the causal mediator of MCD, or the charge hypothesis of glomerular permeability to explain selective proteinuria. Investigators must classify early footprints of glomerular disease based on molecular changes, rather than current morphology based standards. In this context, selective proteinuria could be better explained by looking for uniformity of early molecular changes in the glomerulus in MCD, whereas non-selective proteinuria potentially attributable to presence on non-uniform early changes in other diseases. Unless an objective and goal directed approach is adopted toward developing modern therapeutics, future nephrologists may have to continue to adapt drugs developed for non-kidney indications to treat kidney disease.Sumant S. Chugh is Founder, President and Chief Executive Officer of GDTHERAPYLLC, and filed patents related to the use of Angpt4 mutants (PCT/US2011/039255) and precursors of sialic acid, including ManNAc (PCT/US2011/039058) for the treatment of nephrotic syndrome. SSC may benefit financially from these patents in the future. None of the other authors declared competing financial interests.


In the last decade several groups have determined the key role of hemichannels formed by pannexins or connexins, extracellular ATP and purinergic receptors in physiological and pathological conditions. Our work and the work of others, indicate that the opening of Pannexin-1 hemichannels and activation of purinergic receptors by extracellular ATP is essential for HIV infection, cellular migration, inflammation, atherosclerosis, stroke, and apoptosis. Thus, this review discusses the importance of purinergic receptors, Panx-1 hemichannels and extracellular ATP in the pathogenesis of several human diseases and their potential use to design novel therapeutic approaches. In recent years it has become evident that pannexin (Panx)-1 hemichannels in concert with extracellular adenosine triphosphate (ATP) and purinergic receptors are involved in several physiological and pathological conditions. Recent evidence suggests the participation of Panx-1 hemichannels, extracellular ATP and purinergic receptors in the coordination of events such as cellular activation, apoptosis, stress signals, secretion of inflammatory cytokines, and HIV replication have been explored , although they share no sequence homology. Panx consist of a cytosolic N-terminal domain, four transmembrane domains with two extracellular loops and a cytosolic C-terminal domain  for adenosine and P2 receptors for ATP/ADP receptors  were initially classified as P1 receptors until it was discovered that their agonist was adenosine . Ischemic stroke results from a permanent or transient decrease in cerebral blood flow. This decrease in blood flow is usually caused by the obstruction of a cerebral artery by an embolus or local thrombosis , 15 million people suffer stroke worldwide each year, resulting in 5 million deaths and another 5 million survivors that are permanently disabled  resulted in opening of Panx-1 hemichannels  hemichannels. However, experiments conducted using wild type, Cx43 null, and Panx-1 knocked down astrocytes provided evidence indicating that downregulation of Panx-1 prevented the release of ATP from astrocytes  demonstrated that Panx channels contributed to ischemic brain injury 2+ by activating P2 receptors and P2Y7 receptors  during ischemia have been demonstrated suggesting increased sensitivity of neurons to extracellular concentration of ATP  suggesting that the enhanced lung inflammation observed in these individuals may be associated with ATP dysregulation and purinergic receptor activation  including ulcerative colitis, and Crohn's disease. These diseases are chronic conditions associated with gut dysfunction resulting from alterations in the enteric nervous system leading to severe symptoms  is a heterodimeric transcription factor comprised of α and β subunits , which regulates lipid synthesis. Moreover, eliminating A2B adenosine receptor in the liver of the mouse model increased the levels of SREBP-1 and its downstream targets acetyl coenzyme-A carboxylase- α (ACC) and fatty acid synthase (FAS) resulting in upregulation of lipid synthesis. Resulting in the formation of foam cells and the development of atherosclerotic plaques.  and A2B adenosine receptor which were fed a high fat/high cholesterol diet , which under normal physiological conditions are found in the medial layer of the artery wall. However, under atherosclerotic conditions SMCs lose their contractile element and gain the ability to replicate and migrate into the intima of the arterial wall  membrane and affected intraluminal ER Ca2+ concentration. Using human prostate cancer epithelial cells (LNCaP) and human embryonic kidney cells (HEK-293) they demonstrated that while overexpression drastically reduced intraluminal Ca2+, endogenous Panx-1 depletion using siRNA increased the content of Ca2+ in the ER. This data suggests that Panx-1 hemichannels are not only found on the plasma membrane but also in the ER membrane, and it participates in ER Ca2+ leak and intracellular Ca2+movement. Vanden Abeele et al speculated that the reduced concentration of Ca2+ associated with Panx-1 overexpression could be caused by increase of the BCL-2 family of proteins which plays an important role in the regulation of calcium leak from the ER and is an antiapoptotic protein  were reported in 1981. Since the identification of HIV as the virus responsible for AIDS the countries affected and the numbers of those infected rose to overwhelming numbers. As of 2011, there are 34 million people worldwide living with HIV according to the World Health Organization. A total of 2.5 million new infections were reported in 2011 with 1.7 million deaths related to AIDS.The established model for HIV entry into cells is mediated by the binding of HIV glycoprotein (gp) 120 to the cellular CD4 receptor. This interaction induces a conformational change to allow the glycoprotein to bind to the co-receptors CXCR4 and/or CCR5. The interaction of gp120 with these two host receptors creates a stable attachment between the virus and the cell membrane facilitating successful viral entry into the cell. In the past two decades studies have documented that binding of HIV or gp120 to the cell rapidly increases the intracellular free calcium concentration  and CDOur laboratory recently described a novel role for purinergic receptors in HIV replication in macrophages Figure . We idenPurinergic receptor, Panx-1 hemichannel, and extracellular ATP, ADP, AMP, and adenosine blockers and their potential use as therapeutic agents are under current investigation. Commercially available pharmaceutical adenosine, such as adenocard and adenoscan, are currently used to treat supraventricular tachycardia, is an example of an ion channel targeted treatment  and Brilliant Blue FCF (BB FCF) which are found in most soft drinks. These dyes are shown to block at least P2X7 receptors, and Panx-1 hemichannels respectively (Jiang et al., The P2Y12 platelet receptor plays an important role in the genesis of platelet aggregation (Power et al., Numerous clinical trials are on going using various adenosine receptor agonist and antagonist. The expectation are high and could provide treatments for many physiological and pathological conditions such as lipolysis, renal blood flow, immune function, sleep regulation, angiogenesis, inflammatory diseases, ischemia-reperfusion, and neurodegenerative disorders (Sun et al., Probenecid is a Panx-1 inhibitor, and has been on the market for decades as a treatment for gouty arthritis. High levels of extracellular potassium ion induce inflammasome activation and caspase 1 cleavage in neurons and astrocytes. In addition probenecid has been shown to attenuate the caspase 1 cleavage in cultured neurons induced by extracellular potassium ions (Peng et al., In this review we have discussed the role which purinergic receptors and Panx-1 hemichannels play in the pathogenesis of several human diseases. It is crucial to understand the contribution of these receptors and channels in physiological and pathological conditions, in order to design new and improved therapeutic approaches. The contribution that purinergic receptors and Panx-1 hemichannels play in the HIV viral life cycle has only been recently described and unlocking this relationship could hold the key to the development of new preventative therapies and treatments. Purinergic receptor, Panx-1 hemichannel, and extracellular ATP, ADP, AMP, and adenosine are important modulators of many cellular events and hold great potential in understanding and treating many pathological and physiological conditions. The pathologies discussed in this review contribute to a large number of fatalities worldwide. Although much progress has been made in the advancement of treatments for these pathologies, there are still many avenues, which have not been explored. As more information regarding Panx-1 hemichannels and purinergic receptors emerge, the possibility of new therapeutic opportunities for these pathologies emerges as well.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.