Dopamine is a neuromodulator that is used by neurons in several brain regions involved in motivation and reinforcement, most importantly the nucleus accumbens (NAc). Dopamine alters the sensitivity of its target neurons to other neurotransmitters, particularly glutamate. In addition, dopamine can affect the neurotransmitter release by the target neurons. Dopamine-containing neurons in the NAc are activated by motivational stimuli, which encourage a person to perform or repeat a behavior. Even low alcohol doses can increase dopamine release in part of the NAc. This dopamine release may contribute to the rewarding effects of alcohol and may thereby play a role in promoting alcohol consumption. In contrast to other stimuli, alcohol-related stimuli maintain their motivational significance even after repeated alcohol administration, which may contribute to the craving for alcohol observed in alcoholics. Many substances that relay signals among neurons  are affected by alcohol. Among these, dopamine has received special attention, because several studies have found that alcohol stimulates the activity of a subset of dopamine-releasing neurons and thus enhances dopamine-mediated  in the terminals of their axons. When the dopaminergic neurons are activated, the resulting change in the electrical charges on both sides of the cell membrane  induces dopamine release into the gap separating the neurons  through a process called exocytosis.  To terminate the signaling process, the neurons recapture dopamine through a specific carrier system located on the cell membrane.Dopaminergic neurons originate in three cell groups located in the brain stem. These cell groups are labeled A8, A9, and A10 and correspond to brain regions called the retrorubral field (A8), the substantia nigra pars compacta (A9), and the ventral tegmental area (VTA) (A10). From these cell groups, the axons of dopaminergic neurons extend to discrete regions of the forebrain, forming the following three neuronal systems :nigrostriatal system comprises dopaminergic neurons that originate in the A9 group and terminate in a region called the dorsal striatum. This region, which includes the caudate nucleus and the putamen, is involved in learning to automatically execute complex movements triggered by a voluntary command . The degeneration of dopaminergic neurons in the dorsal striatum causes the motor disturbances that occur in Parkinson’s disease.The mesolimbic system, originates in the A10 and part of the A9 groups. These neurons terminate in the ventral striatum, which includes the NAc and the olfactory tubercle, the septum, the central amygdala, and the bed nucleus of the stria terminalis , which affect most regions of the forebrain.Among the brain areas affected by dopaminergic neurons, the NAc plays a pivotal role. The NAc can be divided into a “shell” region and a “core” region  figure . The sheDopaminergic neurons reach not only the NAc, but also other areas of the extended amygdala as well as parts of the septo-hippocampal system. Consequently, dopamine acts at multiple sites to control the integration of biologically relevant information that determines motivated responding.1-like family, which includes the receptor subtypes D1 and D5, and the D2-like family, which includes the receptor subtypes D2, D3, and D4. When dopamine interacts with these receptors, they activate certain proteins within the cell called G proteins. These proteins, in turn, can affect the activity of channels in the cell membrane that allow the flow of charged particles  into and out of the cell. Changes in the activity of these ion channels make it either easier or more difficult to excite the cell. In addition, G proteins regulate the production of small signaling molecules  within the cell. To affect its target cells, dopamine interacts with specific docking molecules  on the target-cell surface. Dopamine receptor subtypes fall into two families: the DBecause dopamine does not affect the activity of ion channels directly and therefore is unable to excite or inhibit its target cells, it often is not considered a neurotransmitter but is called a neuromodulator . Thus, dTo modulate the responsiveness of neighboring neurons to glutamate, dopamine modifies the function of ion channels in the membrane of the signal-receiving  neuron. The activity of some of these ion channels  depends on the voltage difference, or potential, between the inside and the outside of the cell membrane adjacent to these channels. These channels therefore are called voltage-dependent channels. Through its effects on G proteins, dopamine indirectly modifies the sensitivity with which voltage-dependent channels respond to changes in the membrane potential that occur when glutamate binds to its receptors, which also act as ion channels .1 receptors enhances the excitatory effects that result from glutamate’s interaction with a specific glutamate receptor subtype . Dopamine that has been released from a nerve terminal into the synaptic cleft can travel out of the synapse into the fluid surrounding the neurons and activate these extrasynaptic receptors. Through this mechanism, dopamine modulates the neurotransmitter release that is induced by cellular excitation . For example, activation of some extrasynaptic D1 and D2 families also modulates the release of other neurotransmitters  by nondopaminergic neurons . Accordingly, dopamine’s tonic-nonsynaptic actions could modulate the flow of information across the striatum in the following ways:Activation of some extrasynaptic receptors of the D neurons . Thus, d2-family receptors, which reduce neurotransmitter release, dopamine could reduce signals that represent “background noise,” thereby ultimately sharpening the transmission of information across the striatum.By stimulating D1-family receptors, which enhance neurotransmitter release, dopamine could facilitate the transmission of information from the striatum to other brain areas . These stimuli converge in the A8, A9, and A10 dopaminergic cell groups. From there, the information is passed on to the various brain areas where dopaminergic neurons terminate. Consequently, through the activation of dopaminergic neurons, motivational stimuli can influence the activity of various parts of the brain that might serve different behavioral functions. This mechanism may be one reason underlying the wide range of dopamine’s roles in behavior.The diversity of dopamine’s roles also derives from the fact that dopaminergic neurons belonging to different systems  respond to different kinds of motivational stimuli. Two kinds of motivational stimuli exist: appetitive and con-summatory. Appetitive stimuli serve to attract an individual to a reward  so that the individual can attain it. Consummatory stimuli serve to maintain the individual’s contact with the reward so that the individual can use its biological properties . Some dopaminergic neurons  are activated by both appetitive stimuli  and consummatory stimuli . Conversely, other dopaminergic neurons  are activated only by consummatory stimuli .The different modes of dopaminergic signal transmission also might serve different functions in controlling behavior. For example, dopamine’s phasic-synaptic actions in the NAc shell, which occur in response to natural stimuli such as food, may play a role in motivational learning . This teWhen discussing the consequences of alcohol’s actions on the brain, researchers frequently use terms such as motivation, reinforcement, incentives, and reward. Below are general definitions of these terms.Incentives are stimuli that induce behaviors with the goal of obtaining a reward . Thus, incentives promote responses  necessary for obtaining the reward’s biologically relevant properties . These responses also can include emotions. Incentives themselves have no primary biological properties that could induce motivated behavior. Therefore, incentives obtain motivational properties by being predictably associated with primary biological stimuli.Motivation — a process by which stimuli  come to trigger responses to obtain a reward  or to avoid a punishment  — generally serves to maintain bodily functioning and ensure survival.Motivational learning is the mechanism by which secondary stimuli acquire the ability to elicit behavioral responses. Two types of motivational learning exist: incentive learning and habit learning. In incentive learning, the stimulus acquires the ability to elicit motor as well as emotional responses. In habit learning, the stimulus elicits only physical  responses but is devoid of an emotional content.Reinforcement exists in two types: pavlovian, in which the predictable association of a reward  with a neutral stimulus  strengthens the ability of that stimulus to elicit a response, and operant, in which the stimulus acts as a reinforcer by eliciting a response that enables the subject to obtain (positive reinforcement) or to avoid (negative reinforcement) the same stimulus in the future.Rewards are the goals of motivated behavior. A reward  usually is a complex stimulus having primary  as well as secondary  motivational properties.Stimuli that elicit behavioral responses fall into two categories: primary and secondary. Primary stimuli elicit responses or maintain behaviors without requiring the presence of additional stimuli or prior learning processes. Secondary stimuli depend on primary stimuli to induce a response.However, some food-related stimuli  that activate phasic-synaptic dopaminergic signal transmission in the NAc shell rapidly undergo a form of tolerance  . For exaDopaminergic neurons that relay information to the NAc shell are extremely sensitive to alcohol. For example, in studies performed in rats, alcohol injected into the blood in amounts as low as 2 to 4 milligrams per kilogram of body weight increased dopamine release in the NAc shell and maintained chronic alcohol self-administration . In ratsThe alcohol-induced stimulation of dopamine release in the NAc may require the activity of another category of neuromodulators, endogenous opioid peptides.  This hypothesis is supported by observations that chemicals that inhibit the actions of endogenous opioid peptides  prevent alcohol’s effects on dopamine release. Opioid peptide antagonists act primarily on a brain area where dopaminergic neurons that extend to the NAc originate. These observations indicate that alcohol stimulates the activity of endogenous opioid peptides, leading indirectly to the activation of dopaminergic neurons. Opioid peptide antagonists would interfere with this process, thereby reducing dopamine release.Although numerous studies have attempted to clarify dopamine’s role in alcohol reinforcement by manipulating dopaminergic signal transmission, these investigations do not allow any firm conclusions  stimuli. Orally administered alcohol similarly activates taste receptors, thereby increasing dopamine release in the NAc. In contrast to food, however, alcohol also can modify the function of dopaminergic neurons more directly by entering the brain. Accordingly, oral alcohol administration influences dopamine release in the NAc both through its gustatory properties  and through its direct actions on the brain . Consistent with this hypothesis, two peaks of dopamine release occur in the NAc. The first peak results from the alcohol-related gustatory stimuli; the second results from alcohol’s actions within the brain. Consequently, alcohol-induced direct activation of dopaminergic signal transmission might reinforce the motivational properties of the gustatory stimuli associated with alcohol. As a result of this mechanism, the alcohol-related gustatory stimuli acquire strong incentive properties . Similarly, appetitive stimuli related to alcohol  also acquire incentive properties and promote the search for and consumption of alcohol. Through these complex mechanisms, the alcohol-induced dopamine release activates a secondary reinforcement chain that promotes alcohol consumption.Dopamine release in the NAc shell may be instrumental in the development of alcohol dependence. Psychological dependence on alcohol develops because alcohol-related stimuli acquire excessive motivational properties that induce an intense desire to consume alcohol-containing beverages . As a result of this intense craving, conventional reinforcers  lose their significance and have only a reduced impact on the drinker’s behavior.One mechanism that may be responsible for the abnormal significance associated with alcohol-related incentives is the nonadaptive nature of alcohol-induced stimulation of dopaminergic signal transmission in the NAc. As mentioned previously, enhanced dopamine release in the NAc shell induced by conventional reinforcers  rapidly induces habituation, and repeated presentation of related stimuli no longer induces dopamine release. In contrast, no habituation occurs after repeated alcohol consumption. As a result of the persistent dopamine release in the NAc shell in response to alcohol, alcohol-associated stimuli acquire an abnormal emotional and motivational significance that results in excessive control over the drinker’s behavior. This excessive control constitutes the essence of addiction.Dopaminergic signaling plays a pivotal role in the transmission of motivational stimuli. Dopaminergic neurons primarily affect brain areas involved in mediating the rewarding and reinforcing properties of alcohol and other drugs, most prominently the NAc. Alcohol affects dopamine release in the NAc, not only through its associated gustatory stimuli but also through its direct actions on the brain. The abnormal facilitation of motivational learning that results from alcohol-induced stimulation of dopaminergic signal transmission has been hypothesized to constitute the neurobiological basis of alcohol addiction. Through this mechanism, alcohol-associated stimuli acquire the ability to elicit craving and compulsive alcohol consumption.


N-acetylglucosamine (GlcNAc) and galactose. We have previously shown that GlcNAc-6-O-sulfotransferase (GlcNAc6ST) 1 encoded by Chst2 is an enzyme necessary for the synthesis of GlcNAc-6-sulfated KS chains that are required for neuronal plasticity in the visual cortex of the mouse brain during the critical period, but not in adulthood. Here, we show that GlcNAc-6-sulfated KS recognized by the R-10G anti-KS antibody, of which the minimum epitope structure is Galß1-4GlcNAc(6S)ß1-3Galß1-4GlcNAc(6S), distributes diffusely in neuropils and presents densely in close proximity to the perineuronal region of the perineuronal net (PNN)-positive neurons in the adult visual cortex. Surprisingly, GlcNAc6ST3, which was discovered as an intestinal GlcNAc6ST encoded by Chst5, is a major brain KS sulfotransferase expressed in oligodendrocytes in adulthood. Moreover, we identified an isoform of the protein-tyrosine phosphatase PTPRZ as a R-10G-reactive KS proteoglycan. These results indicate that GlcNAc6ST3 may play a role in synthesis of a component of PNN in the adult brain, and that the KS-modified isoform of PTPRZ encoded by Ptprz1 could be an extracellular molecule associated with PNNs.Keratan sulfate (KS) is a carbohydrate side chain covalently attached to extracellular proteoglycans. KS is composed of disaccharide units of 6-sulfated  CSPGs are also present on neuronal cell surfaces6, forming networks known as perineuronal nets (PNNs)8. PNNs are heterogeneous in composition9, and their formation is concurrent with the termination of the critical period11. We have previously shown that CSPG phosphacan, an isoform of protein-tyrosine phosphatase receptor type Z (PTPRZ) modified with keratan sulfate (KS), is distributed diffusely in the extracellular space and is required for cortical plasticity during the critical period12. In the adult brain, however, distribution of the KS and their proteoglycan core proteins remain largely elusive.Extracellular matrix molecules, such as chondroitin sulfate proteoglycans (CSPGs) and tenascins, interact with each other to fill the extracellular spaces and interpose among neurons and glia in the brainN-acetylglucosamine (GlcNAc) and galactose 14. In the brain, KS chains attach covalently to a core protein mainly through O-mannose-linked glycans in its rather short forms17. Sulfated motifs within KS can be differentially detected with specific antibodies. The R-10G monoclonal antibody recognizes KS structures containing mono-sulfated disaccharides of 6-sulfated GlcNAc and Gal18. The minimum epitope structure is Galß1-4GlcNAc(6S)ß1-3Galß1-4GlcNAc(6S)19. The 5D4 monoclonal antibody detects KS structures composed of di-sulfated disaccharides of GlcNAc and Gal, both 6-sulfated. The Golgi-resident sulfotransferases catalyze C-6 sulfation modifications of GlcNAc and Gal residues of KS14. N-Acetylglucosamine 6-O-sulfortansferase (GlcNAc6ST) transfers a sulfate to GlcNAc. Five and four members of the GlcNAc6ST family have been identified in humans and mice, respectively20. Heparan sulfate 6-O-sulfotransferases that transfer sulfate at C-6 of the N-sulfoglucosamines are distinct from the members of the GlcNAc6ST family21. We have previously generated a GlcNAc6ST1-deficient mouse model22 and reported that deficiency in the GlcNAc6ST1 gene, Chst2, led to a partial reduction of the GlcNAc-6-sulfated KS of phosphacan/PTPRZ in the early postnatal brain23 and in the critical period12. On the other hand, the GlcNAc-6-sulfated KS content was not changed in the adult stage by the deletion of the GlcNAc6ST1 gene12. In order to determine whether other members of the GlcNAc6ST family act on the KS in the adult brain, we analyzed mice deficient in either GlcNAc6ST2 encoded by Chst424, or GlcNAc6ST3, which was discovered initially as an intestinal GlcNAc6ST25 and is encoded by Chst526. Disrupting Chst7 resulted in the GlcNAc6ST4-deficient mice that were analyzed as well. Surprisingly, we found that disruption of GlcNAc6ST3, an intestinal enzyme, eliminated almost all GlcNAc-6-sulfated KS recognized by the R-10G anti-KS antibody in the adult brain, and that GlcNAc6ST3 was selectively expressed in oligodendrocyte precursor cells (OPCs) and the newly formed oligodendrocytes in the adult brain. Moreover, we identified phosphacan/PTPRZ as a major R-10G-positive KS-modified CSPG in the adult brain. The R-10G-positive KS-modified phosphacan/PTPRZ exists diffusely within neuropils and densely in close proximity to perineuronal regions of a subset of PNN-positive neurons in the adult brain cerebral cortex. These results indicate that GlcNAc6ST3 in oligodendrocytes is a major KS enzyme in the adult brain, and that GlcNAc6ST3 may play a role in synthesis of a PNN component, and the KS-modified isoform of PTPRZ could be associated with PNNs.KS is a glycosaminoglycan side chain, consisting of repeating mono- or di-sulfated disaccharides of 23, while GlcNAc-6-monosulfated KS, which is recognized by the R-10G antibody, was expressed at the level comparable to that in the critical period brain12. To confirm that the R-10G recognized molecule is indeed KS-modified, we pretreated the brain samples of adult wild-type (WT) mice with KS-degrading enzymes. Pre-digestion with endo-ß galactosidase and keratanase, which hydrolyze ß-galactosidic linkages in KS chains composed of non-sulfated Gal and 6-sulfated GlcNAc disaccharides ), eliminated the R-10G-reactive KS 19 indicate that the R-10G-positive band is indeed a GlcNAc-6-sulfated KS-modified molecule. Keratanase II hydrolyses 1,3-ß-glucosaminidic linkages to Gal residues in KS composed of Galß1-4GlcNAc(6S) and Gal-6- and GlcNAc-6-sulfated disaccharides ß1-4GlcNAc(6S)). Band intensity was not altered but the apparent molecular weight of the R-10G reactive band at MW > 500 kDa shifted slightly downwards after treatment with keratanase II 8. Intriguingly, dense R-10G staining in the pericellular regions was observed  is hardly detected in the adult mouse brain since PTPRZ-FL is constitutively shed. The ectodomain is proteolytically released and has almost the same structure as phosphacan with a 300 kDa core protein34. Reactivity of the Ptprz-S antibody was negligible in the GlcNAc6ST3-KO sample , keratanase (Pseudomonas sp.), keratanase II (Bacillus sp.), and chondroitinase ABC (P. vulgaris) were from Seikagaku Corporation ; The R-10G anti-GlcNAc-6-sulfated KS antibody19 was from Cosmo Bio ; mouse anti-ß-actin antibody was from Sigma ; horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG1 was from Caltag ; HRP-conjugated goat anti-rabbit IgG (H + L) was from Cell Signaling Technology ; rabbit anti-GLAST and rabbit anti-synaptophysin (SYP) were from Frontier Institute Co., Ltd. ; rabbit anti-parvalbumin (PV) was from Abcam ; Alexa488-conjugated goat anti-rabbit IgG (H + L), Cy™3-conjugated goat anti-mouse IgG1, and HRP-conjugated goat anti-mouse IgG (H + L) were from Jackson ImmunoResearch Laboratories ; Fluorescein isothiocyanate (FITC)-conjugated Wisteria floribunda lectin (WFA) was from Vector Laboratories, Inc. ; NeuroTrace™ Fluorescent Nissl Stain was from Thermo Fisher Scientific .The following materials were obtained commercially from the indicated sources. Endo-ß-galactosidase (Chst2−/−), and GlcNAc6ST2-KO (Chst4−/−) mice47, and GlcNAc6ST3-KO (Chst5−/−) mice26 were maintained on a C57BL/6 J genetic background. GlcNAc6ST4-KO (Chst7−/−) mice were generated by an in vitro fertilization at the Nagoya University Animal Care Department by obtaining the frozen sperm of the Chst7−/− mice originated from the trans-NIH Knockout Mouse Project (KOMP). Genomic organization and targeting strategy of the mouse Chst7 locus is shown in Fig. Chst7 allele and yielded a PCR product of 0.44 kb. Chst7-F1 and Chst7KO-R1 primer set amplified the sequence of the KO cassette-inserted allele and yielded a PCR product of 0.25 kb. Males and females of all genotypes at 2- to 6-month old were used for experiments. All mice were maintained under controlled SPF environmental conditions and provided with standard nourishment and water in the animal facilities of Nagoya University Graduate School of Medicine and Research Institute of Environmental Medicine. All experiments were approved by the Animal Research Committee of Nagoya University, and performed in accordance with the guidelines of Nagoya University.C57BL/6 J mice were purchased from SLC Inc. . GlcNAc6ST1-deficient (KO). Brains were dissected out and divided into sagittal parts. Regional parts of hemi-brains, namely, brainstem, thalamus, hippocampus, cerebellum, olfactory bulb, and cerebral cortex, were separated on ice, snap-frozen, and then stored at −80 °C for biochemical analysis. Hemi-brains were post-fixed overnight in phosphate buffer (PB) containing 4% paraformaldehyde, equilibrated into 30% sucrose in PBS and then embedded into Tissue-Tek®  for frozen sectioning.g for 15 min at 4 °C. The supernatants were used as the 1% Triton-soluble fraction. The protein concentration was measured by the Bradford method. For pretreatment, 1% Triton-soluble fractions were heated for 10 min at 95 °C. Supernatants were collected by centrifugation at 10,000 × g for 3 min at 4 °C. The supernatants were treated with Endo-ß-galactosidase (10 µU/µl), keratanase (1 mU/µl), or keratanase II (50 µU/µl) at 37 °C overnight. In the case of treatment with chondroitinase ABC (1 mU/µl), the reaction time was 3 h. The enzymatic reaction was stopped by heating the samples at 95 °C for 5 min.Snap-frozen brain samples (~20 mg) were homogenized with a Dounce homogenizer in 600 µL (30 volumes of the tissue weight) of cold Tris-buffered saline (TBS) containing 1% Triton X-100 (w/v) and cOmplete™ protease inhibitor cocktail (Roche). The homogenized samples were placed on ice for 30 min. Supernatants were collected by centrifugation at 10,000 × 34 in 5% skim milk/TBS-T overnight at 4 °C. Membranes were washed with TBS-T and incubated for 30 min at room temperature with HRP-conjugated secondary antibodies. Bound antibodies were detected with a Novex ECL Chemiluminescent reagent kit (Thermo Fisher Scientific) and an Amersham Imager 600 . Densitometry analysis of immunoreactive bands was performed with the ImageJ software (http://imagej.nih.gov/ij/) .The proteins (20 µg) were separated using 7.5% polyacrylamide gel electrophoresis  and blotted onto a polyvinylidene difluoride (PVDF) membrane . The membrane was blocked with 5% skim milk in TBS containing 0.1% Tween-20 (TBS-T) for 1 h at room temperature and then incubated at 4 °C overnight with primary antibody: R-10G anti-KS antibody (1:200 dilution) or the rabbit polyclonal anti-Ptprz-S antibody (1:500 dilution)The proteins (200 µg) in the 1% Triton-soluble fraction of the cerebral cortex were mixed with a complex of the R-10G anti-KS antibody and Protein G Dynabeads™ (Thermo Fisher Scientific) in PB containing 0.02% Tween-20 (PB-T) for 30 min at room temperature. The immunocomplexes bound to the Protein G Dynabeads™ were isolated with the DynaMag™ -2 Magnet (Thermo Fisher Scientific). Pretreatment of the samples with chondroitinase ABC was performed in the case of immunoprecipitation followed by the R-10G immunoblot, and molecular identification with liquid chromatography-tandem mass spectrometry (LC-MS/MS).48. The resultant digested peptides were reconstituted in 0.1% formic acid and analyzed by a Thermo Orbitrap Elite mass spectrometer (Thermo Fisher Scientific). The peptides were directly infused into the ESI source through a packed nano-capillary column  equilibrated in 0.1% formic acid at a flow rate of 300 nL/min, and were sequentially eluted with an acetonitrile gradient of 0–30% for 10 min, 30–80% for 2 min, followed by an 8-min hold at 80%. The spectrometer was operated in a data-dependent mode using the normalized collision energy of 30. The temperature of the ion transfer tube was set at 250 °C. The spray voltage was at 2.0 kV. The full mass spectra were acquired using an m/z range of 350–2000. The resultant MS and MS/MS data were searched against the NCBI database using the Mascot ver.2.5.1  with Proteome Discoverer software (Thermo Fisher Scientific).For LC-MS/MS analyses, polyacrylamide gel electrophoresis separation and an in-gel trypsin digestion of the R-10G immunoprecipitated glycoproteins with MW > 300 kDa were performed as described previouslyFrozen brain tissues were cut into 10-µm-thick sections on a cryostat and collected on MAS-coated glass slides . Sections were air-dried for 30 min, rinsed with PBS to remove O.C.T. compounds, and then blocked in PBS containing 5% normal goat serum and 0.3% Triton-X 100 for 1 h at room temperature. Sections were incubated with R-10G (1:100 dilution), and rabbit anti-GLAST , rabbit anti-SYP (1:200), rabbit anti-PV (1:200), or FITC-conjugated WFA (1:100) in PBS containing 0.03% Triton-X 100 at 4 °C overnight. Sections were washed with PBS and then incubated with Cy3-anti-mouse IgG1 (1:400 dilution) and Alexa488-conjugated goat anti-rabbit IgG (H + L) (1:500) for 30 min at room temperature. After washing with PBS, sections were incubated with Hoechst 33342 solution  for 5 min at room temperature for staining cell nuclei, or NeuroTrace™ 435/455 blue fluorescent Nissl stain for visualizing neurons. Stained sections were mounted in FluorSave™ Reagent . Signals were visualized and captured by a fluorescent microscope  at the same exposure setting for each antibody, or a confocal microscope.Cryostat-cut brain sections (10-μm-thick) from adult mouse brains were co-stained as described above. The signals were visualized with a total internal reflection fluorescence/confocal laser scanning microscope . The Z-step scans were recorded . The images were 3D-reconstructed and analyzed by NIS-Elements analysis software (Nikon).49. Comparison of their transcription profiles in various cell types of the brain was performed with an RNA-Seq transcriptome platform (http://web.stanford.edu/group/barres_lab/brain_rnaseq.html). A value of fragments per kilobase of transcript sequence per million mapped fragments (FPKM) of ~0.04 was determined as a threshold for minimum gene expression49.Data pertinent to the genes of GlcNAc6STs and phosphacan were mined from a published RNA sequencing (RNA-Seq) analysis of purified neurons, oligodendrocyte precursor cells, newly formed oligodendrocytes, myelinating oligodendrocytes, astrocytes, microglia, and endothelial cells from adult mouse brainsP < 0.05. All data are means ± SD unless otherwise noted.Values were analyzed via the one-way analysis of variance with Tukey’s test Fig.  by usingSupplementary InformationTable S1


Epithelioid cell granuloma with caseating necrosis is a typical pathological finding in tuberculosis. While specific inflammation, including that related to tuberculosis, can induce caseating granuloma formation, there have been very few reports on the induction of caseating granuloma by non-specific inflammation. Chronic periapical periodontitis is usually related to bacterial biofilm formation as well as fungal or viral infection in the periapical lesion. However, it is difficult to eliminate these extraradicular pathogenic microbes by normal endodontic therapy alone, and more invasive surgical removal is almost always required.Here we describe the case of a 30-year-old Japanese woman who had suffered from dull pain related to periapical periodontitis for approximately 10 years. Although the causal tooth had been previously extracted at the Department of Oral Surgery of another hospital in 2015, inflammation of the surrounding tissue had not abated. She was referred to our hospital in May 2016 and underwent surgical debridement via an intra/extraoral approach under general anesthesia. A caseating granuloma accompanied by a small amount of fungi was histopathologically confirmed in the excised specimen. Her inflammation has not been exacerbated since the operation.This is the first report in which non-specific inflammation is shown to induce caseating granuloma arising in the jaw. Our report also highlights the importance of sufficient root canal treatment during the first stage of the procedure. Epithelioid cell granuloma accompanied by Langhans multinucleated giant cells and enclosing caseating necrosis is a typical pathological finding in tuberculosis. In the head and neck area, tuberculosis commonly develops in the oral mucosa, especially the gingiva, and in the lymph nodes. Tubercles are rarely seen in the jaw, as tuberculous osteomyelitis occurs in < 2% of patients with skeletal tuberculosis . HoweverAmong the causes of persistent periapical periodontitis is endodontic failure due to defective instruments, perforations, overfilling, underfilling, ledges, and root canal therapies with iatrogenically altered root canal morphology . In the Here we describe a case of long-term periapical periodontitis in which overextended gutta-percha possibly induced the formation of an epithelioid cell granuloma with caseating necrosis.A 19-year-old Japanese woman underwent root canal treatment on tooth #47 by a general practitioner in 2005, but dull pain persisted after. Tooth #48, which was horizontally embedded, was extracted in August 2010, but the pain continued. In August 2015, she presented at the Department of Oral Surgery of another hospital with a purulent discharge from the pocket of tooth #47. Intraoral radiography showed insufficient root canal filling in the distal root and a foreign body, suspected to be extruded gutta-percha, outside the apex of the mesial root of tooth #47, accompanied by bone absorption around both roots Fig. . ComputeSurgical debridement under general anesthesia was performed. Via the submandibular region, an incision was made in the thickened periosteum at the inferior border of the mandible, after which the lingual periosteum was separated from the mandible Fig. . Tooth #On histopathologic examination, a large epithelioid cell granuloma with central caseating necrosis was observed in the dense fibrous tissue Fig. . The graEpithelioid cells are activated macrophages with a shape similar to that of epithelial cells. Epithelioid cell granuloma may occur at sites of intense inflammation and may be caseating or non-caseating. The caseating type occurs in the setting not only of specific inflammation, including tuberculosis and syphilis, but also of non-specific inflammation. The non-caseating type is seen in patients with sarcoidosis and leprosy, among other diseases.Our patient did not present with the clinical findings typical of tuberculosis, that is, cough, sputum, and lymphadenopathy. She also had no abnormal findings on her chest X-ray and blood test. The pain had persisted for approximately 10 years, and there was no family history of tuberculosis. Acid-fast bacilli were not detected. Together, these findings ruled out tuberculosis. Syphilis was excluded by blood test.et al. [Salmonella typhi, which was healed by saucerization. Assari et al. [There are very few reports in which a caseating epithelioid cell granuloma was induced by non-specific inflammation. Szilagyi et al.  reportedi et al.  describePorphyromonas gingivalis, Tannerella forsythensis, and Prevotella intermedia are often detected in periapical biofilms [Candida albicans as the most commonly isolated species [In our patient with clinically diagnosed osteomyelitis of the mandible we chose treatment via surgical debridement. Osteomyelitis in the head and neck is induced by various factors. The incidence of osteomyelitis in the mandible and maxilla of patients with odontogenic predisposing factors is reported to be 22% and 30%, respectively . Odontogbiofilms , but man species . The pre species , 7. Curr species .In our patient, periapical periodontitis might have induced the epithelioid cell granuloma with caseating necrosis, given the chronic dull pain that developed even after the root canal treatment of tooth #47. A dental X-ray before the extraction of tooth #47 showed insufficient root canal filling. We previously reported that Garrè’s osteomyelitis induced by periapical infection could be healed by a root canal procedure . Had ourThis is the first report of a patient with neglected periapical periodontitis that may have induced an epithelioid cell granuloma with caseating necrosis in intra/extra mandibular bone. Treatment consisted of surgical enucleation but also required extraction of the vital adjacent tooth. In this patient, endodontic failure at the first stage made it impossible to control the inflammation locally, whether by retreatment, apicectomy, or even extraction, and surgical debridement under general anesthesia was therefore inevitable. This case well demonstrates the importance of appropriate and sufficient root canal treatments during the first stage of the procedure.


Ganoderma lucidum and four commercial mushrooms, that is, Pleurotus ostreatus, Volvariella volvacea, Hericium erinaceus, and Lentinus edodes from Pakistan were screened for their biological activities such as anticancer, antityrosinase, anti‐α‐glucosidase, and antithrombotic activities from their methanol, ethanol, and water extracts. Enzyme inhibition assay showed that selected mushrooms are potent inhibitors with %age inhibition ranging from 19.00% to 80.91%, and 32.85% to 83.38% for tyrosinase and α‐glucosidase, respectively. The best tyrosinase inhibition was shown by P. ostreatus whereas L. edodes was found best as α‐glucosidase inhibitor. These mushrooms were tested against cancer cell lines including HT‐29 colon and H‐1299 lungs carcinoma cell lines. G. lucidum showed 29% and 24% viability of cells against HT‐29 and H‐1299 cell lines, respectively. This antiproliferative effect was in dose‐dependent manner, and the maximum inhibition was observed at 200 μg/ml. Mushrooms extracts were also found effective against clot lysis. The percentage of clot lysis was in the range of 27%–29%. The research would provide knowledge to the people of Pakistan about the importance of locally available commercial mushrooms and wild mushrooms for health improvement and prevention against different kinds of diseases.Mushrooms have been accepted as nutraceutical foods because of their high nutritional and functional values. They have also gained interest due to their medicinal properties, economic importance, and organoleptic merit. In this study, wild  Although the improvements brought by technology has made life relaxed to the people, but many are still looking for improved herbal substitutes that are proved to be more effective in their utmost natural form , 148,041 people were reported to have cancer in 2012 among which death cases were about 101,113 in Pakistan , cardiovascular diseases (CVDs)  are the causes of approximately 30% of deaths worldwide  P. Karst., was isolated from the stem of Salmalia malabarica plant collected from Jinah garden, Faisalabad. Commercial locally cultivated Pleurotus ostreatus (Jacq. Ex. Fr.) Kumm., Volvariella volvacea  Sing., were collected from Horticulture Department, University of Agriculture Faisalabad, and exotic commercially available mushrooms Lentinus edodes (Berk.) Sing. and Hericium erinaceus (Bull.) Pers. (imported from China) were collected from local market. All the selected mushrooms were collected in dry form. Taxonomic identification was made by Prof. Dr. M. Asif Ali from medicinal mushroom lab, Institute of Horticultural Sciences, University of Agriculture Faisalabad, Pakistan. The specimen of each species was grounded in a domestic blender and reduced to fine dried powder and stored at 4°C before the extractions.The wild locally grown 2.2The selected mushrooms were extracted in methanol, ethanol, and water according to the method given by Gangadevi, Yogeswari, Kamalraj, Rani, and Muthumary  with sli2.3The in vitro cell proliferation assay was conducted as described by Jeff et al. . The numA and B are the average numbers of viable tumor cells for the control and samples, respectively,  test compound, followed by the addition of 10 μl (0.057 units) enzyme. The contents were mixed, preincubated for 10 min at 37°C, and preread at 400 nm. The reaction was initiated by the addition of 10 μl of 0.5 mm substrate (p‐nitrophenyl glucopyranoside). Acarbose was used as a positive control. After 30 min of incubation at 37°C, absorbance was taken at 400 nm using microplate reader (BioTek‐USA). The percent inhibition was calculated by the following equation: The α‐glucosidase inhibition activity was performed according to the slightly modified method of Kwon, Apostolidis, and Shetty  and Dong50 values  of compounds were calculated using EZ‐Fit Enzyme Kinetics Software .IC2.5The antityrosinase effect of mushrooms was determined by calculating the hydroxylation of L‐tyrosine to L‐DOPA. Inhibition assay was conducted in 96‐well microplates, a spectrophotometer reader was used to determine the absorbance at 490 nm. Kojic acid was used as a positive control.  was suspended in 1 ml dimethylsulfoxide (1%), and the suspension was shaken vigorously on a vortex.Blood samples of different healthy volunteers were collected from different hospitals and laboratories of Faisalabad, Pakistan. Venous blood was drawn from healthy human volunteers without a history of oral contraceptive or anticoagulant therapy and irrespective of gender. Blood (500 μl) was transferred to the previously weighed microcentrifuge tubes.2.6.2Preweighed microcentrifuge tubes which contain blood were incubated at 37°C for 45 min. Blood clot was formed at the bottom of each centrifuge tube. The serum was removed without disrupting the clot. Clots were weighed again to calculate weight of clot before lysis. Weight of clots (Wc) was determined by taking the difference of weight of microcentrifuge tubes (Wm) containing clot and weight of empty microcentrifuge tubes (We).c = wm–weW2.6.3Each mushroom extract (100 μl) was added in tubes, where streptokinase and distilled water were applied as positive and negative controls, respectively. All the microcentrifuge‐tubes were again incubated at 37°C for 90 min. for clot lysis. Then, tubes were inverted and left overnight. Microcentrifuge tubes were taken out of the incubator and the fluid obtained after lysis along with the applied agents  was removed carefully. Tubes were weighed to calculate the weight of clot after lysis. The weights of clots were determined by taking difference between weights of clot after lysis (W1) and weight of empty tubes (We).c = Wl–WeWThen, percentage of clot lysis activity of different mushrooms extracts was determined by the difference between weight of clots before (Wb) and after lysis (W1) dividing by weight of clot before lysis and multiplied by 100.2.6.4Mushrooms extracts of different concentrations  and incubated at different time intervals , and 30, 60, and 100 μl of each concentration  of mushroom extract were used to determine the effect on thrombolysis.2.7The results obtained were presented by means ± standard deviation.33.1Cancer diseases are one of the main causes of death worldwide  and currently being utilized worldwide as dietary supplement  among the selected commercial mushrooms followed by L. edodes (42.82%) whereas the H. erinaceus and V. volvacea exhibited less activities. Similarly, the IC50 values were lower for P. ostreatus, 34.78 μM as shown in Table In the synthesis of melanin pigments, tyrosinases are responsible for coloring hairs, skin, and eyes and also for the treatments of some dermatological hyper pigmentation illness connected with overproductions of melanin , cardiovascular diseases (CVDs)  are the causes of approximately 30% of deaths worldwide  at concentrations of 0.3%, 0.6%, and 1% were 13.8%, 20.9%, and 27.4% respectively, and for H. erinaceus (ethanol) at 0.3%, 0.6%, and 1% were 16.7%, 18.4%, and 27.1%, respectively. The percentage values of clot lysis at different concentrations were ranged from 13% to 27% showing that percent lysis is directly proportional to concentration and the maximum activity was shown at 1% concentration. The results are presented in Table The percentage values of clot lysis of mushrooms extracts were directly proportional to concentrations, time of incubation, and amount of extract. The percentage values of clot lysis for Lentinus edodes (ethanol) at different amounts of 30, 60, and 100 μl were 14.3%, 16.5%, and 18.2%, respectively. For G. lucidum (ethanol) at 30, 60, and 100 μl, the values were 7.4%, 12.6%, and 17.1%, respectively, the percentage of clot lysis at different amounts were ranged from 7% to 18%. Ethanolic extracts of H. erinaceus showed the percentage values of clot lysis 11.11%, 13.0%, and 16.9% at 30, 60, and 100 μl, respectively. Increase in percentage clot lysis was observed by increasing amount of extract.H. erinaceus (ethanol) recorded at different times of incubation at 30, 60, and 90 min were 1.326, 1.874, and 1.953 nm, respectively. The absorbance of clot lysis at each time of incubation differed from each other. In case of V. volvacea (ethanol), the absorbance values of clot lysis were 0.596, 1.276, and 1.568 nm, respectively, at 30, 60, and 90 min of incubation (Table The absorbance for L. edodes and H. erinaceus extracts have significant thrombolytic activity as compared to the local cultivated mushrooms P. ostreatus. Wild G. lucidum also showed moderate thrombolytic potential.Up to our knowledge, no study has been conducted on thrombolytic potential of these selected mushrooms. This study showed that The authors declare that they do not have any conflict of interest.This study does not involve any human or animal testing.


Since the onset of land application of poultry litter, transportation of microorganisms, antibiotics, and disinfectants to new locations has occurred. While some studies provide evidence that antimicrobial resistance (AMR), an evolutionary phenomenon, could be influenced by animal production systems, other research suggests AMR originates in the environment from non-anthropogenic sources. In addition, AMR impacts the effective prevention and treatment of poultry illnesses and is increasingly a threat to global public health. Therefore, there is a need to understand the dissemination of AMR genes to the environment, particularly those directly relevant to animal health using the One Health Approach. This review focuses on the potential movement of resistance genes to the soil via land application of poultry litter. Additionally, we highlight impacts of AMR on microbial ecology and explore hypotheses explaining gene movement pathways from U.S. broiler operations to the environment. Current approaches for decreasing antibiotic use in U.S. poultry operations are also described in this review. Antimicrobial compounds and antibiotics in U.S. broiler (meat chicken) operations have widely been used to treat and prevent bacterial, protozoal, and fungal pathogens that sicken or kill birds, as well as promote growth . ConsideThe first use of antibiotic drugs in poultry can be traced back to 1946  and firsAccording to the World Health Organization (WHO), antimicrobial resistance (AMR) is defined by “an increase in the minimum inhibitory concentration of a compound for a previously sensitive strain” . There aWhile specific links between antibiotic-use in animal agriculture and human health have been debated , one conFinally, the U.S. is the world’s largest poultry producer with over 9 billion broilers produced annually, with roughly 45% of broilers being produced in 4 mid-south states . Poultry litter is a combination of bedding material, poultry excreta, spilled feed and feathers and is produced in significant quantities. By some estimates, nearly 13 million Mg (14 million tons) of broiler litter is produced on U.S. poultry farms annually . Consequ6–109 bacterial cells of 103–106 different bacterial species (Streptomyces spp.) or fungi , studies have shown that commensal (non-pathogenic) and pathogenic microorganisms share resistance genes with soil communities. Specifically, contact of antimicrobial compounds may stimulate bacterial stress response, which can result in increased mutation rates in co-dispersed bacteria, with co-selection amplifying this effect; thus allowing clustering of ARG . For exavery day . This isvery day .The pathogenic bacteria pathway from the animal through the environment is complicated and more longitudinal studies are needed to follow AMR genes through agricultural systems . These cThe role of the environment as a transmission route for bacterial pathogens has long been recognized, often associated with fecal contamination of water or organic fertilizer applications . DependiSeveral research efforts have been conducted for testing sources of AMR pathogenic bacteria and the transmission route from the chicken flock, to processing, and the larger environment . NumerouEnterococci , was introduced via poultry manure into soil. Approximately 6.9 kg per 1000 kg live weight per day is produced for a typical broiler operation, or 0.6–1.8 Mg per 1,000 broilers per flock  supplements for reducing poultry pathogenic bacteria colonization. Extensive research in the last 2 decades has identified a plethora of compounds with antimicrobial efficacy . Compoun poultry , b. Mediand feed . Similaremulsion  thyme, aemulsion  in poultIdentifying feasible management practices is one of the objectives of the USDA Action Plan to control AMR development in animal agriculture . EstabliCampylobacter than from conventional grown farms  as it relates to transfer  of genetic resistance is a multifaceted issue and widely considered a key challenge facing agriculture.A major challenge facing microbiologists is to track dissemination of resistance genes in poultry production systems and identify reservoirs of resistance genes. Understanding factors that drive selection and dissemination of environmental antibiotic resistance, as well as mitigation strategies that will reduce the environmental dissemination of ARG. Future improvements in monitoring AMR movement in surface water from land-applied poultry litter will be critical to prevent the spread of resistance genes in the environment.The pathogenic bacteria pathway from animals through the environment is complicated and more research is needed to follow AMR genes through these systems using the One Health Approach. Finally, owing to consumer demand for antibiotic-free meat products, much research has been done on promising antibiotic replacements . However, further research is needed on their efficacy and influence on AMR gene movement from farm-field.YY, AA, KC, CW, and AU assisted in writing sections of the manuscript, as well as conceived of the outline and overall direction of this manuscript. PO, SR, JD, and PM provided final edits and direction on the current state of the literature, as well as guidance on soil and poultry microbiology.Mention of tradenames or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.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.


Ganglioneuromas (GN) are rare, benign tumors derived from neural crest cells. They are in the same family of neuroblastic tumors that includes the intermediate ganglioneuroblastoma (GNB) and the malignant neuroblastoma (NB), each of which carries a different prognosis based on tumor histopathology. GNs are generally asymptomatic and usually found incidentally when the tumor becomes palpable or has grown large enough to exert mass effect on adjacent structures. Unlike their malignant counterparts, GNs are rarely hormonally active and usually do not exhibit systemic metabolic activity. We present a case of an adolescent female with a pelvic tumor that was found to be a purely dopamine-secreting GN. Resection resulted in sudden dopamine withdrawal and unexpected severe emotional lability post-operatively.A 16-year-old female presented with a history of increasingly irregular menses over the past year and was found to have an 8-centimeter pelvic tumor. Subsequent work up revealed the mass to be solely dopamine secreting. The tumor was excised without preoperative hormonal blockade. Post-operatively, the patient developed severe emotional lability and symptoms of depression, likely related to the acute withdrawal of circulating dopamine.Ganglioneuromas are rarely metabolically active. However, a preoperative endocrine workup should be done to rule out other more commonly hormonally active tumors such as neuroblastomas, pheochromocytomas, and paragangliomas. If isolated dopamine secretion is found, hormonal blockade is not required preoperatively and operative manipulation and removal should be considered safe. However, one should anticipate potential emotional and psychiatric issues post-operatively due to the acute withdrawal of circulating dopamine. Due to 2A 16-year-old girl with a history of irregular menses for one year presented to the surgical clinic after a screening ultrasound revealed a pelvic mass. She subsequently underwent a pelvic MRI which suggested that this 8-centimeter mass was distinct from the ovary and therefore the possibility of a paraganglioma or neuroblastic tumor was raised . The patPostoperatively, the patient was monitored in the ICU. Overnight, she had severe anxiety and crying episodes requiring dexmedetomidine infusion, which was eventually converted to oral lorazepam. Her pain was well-controlled and she tolerated a regular diet on postoperative day one, but remained hospitalized for an additional two days predominantly to control her anxiety. By postoperative day three, the patient no longer required lorazepam and was able to be discharged home. In follow up, her mood had returned to baseline and she has had no evidence of recurrence in nearly two years. Pathologic evaluation confirmed the tumor to be a ganglioneuroma, maturing subtype.3Ganglioneuromas are rare neural crest tumors that represent 6.4–20% of all neuroblastic tumors and have an overall reported incidence of 1 in 1,000,000 . Many ofFew ganglioneuromas are hormonally active, a characteristic more often associated with neuroblastomas, pheochromocytomas, and paragangliomas . Those tGanglioneuromas can also secrete dopamine, either exclusively or in addition to epinephrine and norepinephrine. These are exceptionally rare as dopamine-secreting tumors are generally extra-adrenal pheochromocytomas and a review of the existing world literature highlights only 17 published cases and reports of dopamine producing ganglioneuromas to our knowledge . Of thesThis poses significant difficulty in appropriate diagnosis given the rarity of this type of tumor and the relative asymptomatic clinical presentation. When epinephrine and norepinephrine are present, the symptoms typically found include unexplained hypertension, facial flushing, palpitations, or diaphoresis ,10. HoweImaging that may help distinguish between active and inactive neuroblastic masses includes the nuclear medicine study measuring metaiodobenzylguanidine (mIBG) uptake, which has increased uptake when catecholamines, HVA, or VMA are present. However, mIBG studies may further complicate the diagnosis as neuroblastomas and pheochromocytomas also have increased uptake of mIBG and uptake is not always present in ganglioneuromas . UltimatThough dopamine-secreting ganglioneuromas may be clinically silent compared to those that secrete more vasoactive compounds, it remains important to appropriately diagnose a dopamine-secreting tumor. Dopamine exerts an antiadrenergic effect and prevents the vasoconstriction promoted by the other catecholamines, thereby limiting the hypertension that would other be present if there was a mixed-hormone secreting mass . RemovalManagement of ganglioneuromas is traditionally limited to surgical excision without adjuvant or neoadjuvant chemoradiation ,5,7,8. Oth reported case to our knowledge of a ganglioneuroma that exclusively produced dopamine. She did not require pre-operative alpha or beta blockade and was managed with a laparoscopic-converted-to-open excision of the pelvic ganglioneuroma. Acute withdrawal of dopamine may have contributed to her emotional lability and depressive symptoms post-operatively. She is currently doing well and will be monitored closely for any recurrence.Our patient represents the 64Though uncommon, ganglioneuromas can be hormonally active and can secrete a variety of catecholamines that require appropriate peri-operative management prior to surgery. This complicates diagnosis as these neuroblastic tumors must be distinguished from pheochromocytomas, neuroblastomas, and paragangliomas. Therefore, appropriate work up including imaging and endocrine studies should be performed to determine management and prognosis. As our case study shows, diagnostic consideration should be made to specifically screen for dopamine secretion as isolated production is rare, but this stimulus may be attributable to post-resection psychiatric symptoms. Management can include incomplete surgical resection to reduce morbidity with close follow up and monitoring for progression.No study sponsors.The case report has been approved for publication and reporting by the Institutional Review Board for our institution.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.Louis Chai: Investigation, resources, data curation, writing – original draft, writing – review and editing, visualizationSean Ciullo: Conceptualization, investigation, resources, data curation, writing – original draft, writing – review and editing, visualization, supervision, funding acquisition. Pre- and post- operative care, operative surgeon.Rajeev Prasad: Investigation, resources, conceptualization, data curation, writing – original draft, writing – review and editing, visualization, supervision, funding acquisition. Pre- and post-operative care, operative surgeon.Not a research study.Rajeev Prasad.The authors of this paper have no conflicts of interests or disclosures to be reported.Not commissioned, externally peer-reviewedNo conflicts of interests from any author.


Enterocytozoon hepatopenaei (EHP) is a newly emerged microsporidian parasite that causes retarded shrimp growth in many countries. But there are no effective approaches to control this disease to date. The EHP could be an immune risk factor for increased dissemination of other diseases. Further, EHP infection involves the absence of obvious clinical signs and it is difficult to identify the pathogen through visual examination, increasing the risk of disease dissemination. It is urgent and necessary to develop a specific, rapid and sensitive EHP-infected shrimp diagnostic method to detect this parasite. In the present study, we developed and evaluated a rapid real-time loop-mediated isothermal amplification  for detection of EHP.A rapid and efficient real-time LAMP method for the detection of EHP has been developed. Newly emerged EHP pathogens in China were collected and used as the sample, and three sets of specificity and sensitivity primers were designed. Three other aquatic pathogens were used as templates to test the specificity of the real-time LAMP assay. Also, we compared the real-time LAMP with the conventional LAMP by the serial dilutions of EHP DNA and their amplification curves. Application of real-time LAMP was carried out with clinical samples.Positive products were amplified only from EHP, but not from other tested species, EHP was detected from the clinical samples, suggesting a high specificity of this method. The final results of this assay were available within less than 45 min, and the initial amplification curve was observed at about 6 min. We found that the amplification with an exponential of sixfold dilutions of EHP DNA demonstrated a specific positive signal by the real-time LAMP, but not for the LAMP amplicons from the visual inspection. The real-time LAMP amplification curves demonstrated a higher slope than the conventional LAMP.In this study, pathogen virulence impacts have been increased in aquaculture and continuous observation was predominantly focused on EHP. The present study confirmed that the real-time LAMP assay is a promising and convenient method for the rapid identification of EHP in less time and cost. Its application greatly aids in the detection, surveillance, and prevention of EHP. Enterocytozoon hepatopenaei (EHP) is the microsporidian parasite that causes hepatopancreatic microsporidiosis in shrimp  to develop a real-time loop-mediated isothermal amplification  for detecting EHP. The real-time LAMP assay is a more sensitivity and accurate diagnostic method, which enables the detection of low-level infections and infection in the host. This assay remained a critical tool to detect EHP in shrimps.Four EHP positive shrimp samples are naturally infected were collected from the shrimp ponds in Zhanjiang, Guangdong, China in February 2017. All the positive samples were confirmed by the state key laboratory for crustacean quarantine. Clinical samples were collected from the shrimp ponds in Zhanjiang, Guangdong, China. Then, the dissected hepatopancreatic tissues of the shrimp were taken and preserved separately using 95% ethanol with more than three times the volume. The White spot syndrome virus (WSSV), Macrobrachium rosenbergii Nodavirus (MrNV), and Infectious hypodermal and hematopoietic necrosis virus (IHHN) used in the present study was provided by the research teams at state key laboratory for crustaceans quarantine.0 to 10−6) were prepared using Tris buffer , and then stored at 20 °C until further use.The hepatopancreatic tissues were rinsed under sterile water to remove ethanol. This was followed by mixing of 50 mg–1 g of shrimp hepatopancreatic tissues with two times the volume of lysis buffer  and sterile water) at room temperature for 5 min, and then lysed at 95 °C for 5 min. The mixture was transferred to the DNeasy Mini spin column and centrifuged for 1 min. Serial dilutions of EHP DNA (10https://primerexplorer.jp/elamp4.0.0/index.html). These primers were synthesized by Sangon Biotech Co., Ltd .The real-time LAMP primers targeting EHP were selected from the established methods for this study . The rea4)2SO4, two mM MgSO4, 0.1% of Triton X-100, 1.4 mM dNTPs  each, and sterile water was used to make up to 25 μl. The fluorescent signals were observed automatically by StepOne Real-Time PCR System .The real-time LAMP reaction was performed in a total volume of 25 μl, including 0.5 μl (8U) of Bst DNA Polymerase Large Fragment , four μM of SYTO-9 , one μl EHP-OF & EHP-OB (0.2 μmol/l), one μl EHP-IF & EHP-IB (1.6 μmol/l), one μl EHP-LF & EHP-LB (0.8 μmol/l), one μl of EHP template DNA, and 13 μl of real-time LAMP buffer. The real-time LAMP buffer contains 20 mM Tris–HCl, 10 mM KCl, 10 mM (NH4)2SO4, two mM MgSO4, 0.1% of Triton X-100, primer mix , two μl EHP template DNA, 1.4 mM dNTPs each), and sterile water was used to make up to 25 μl. The reaction was carried out at 60 °C for 45–90 min.The LAMP reaction was performed in a total of 25 μl volume, including 0.5 μl (8U) of Bst DNA Polymerase Large Fragment , four μM of SYTO-9 , one μl, 12.5 μl of LAMP buffer  were used to be a positive sample, and ddH2O was used as a negative control. The real-time LAMP the fluorescence signal date was obtained from StepOne Real-Time PCR System.The sensitivity of real-time LAMP procedure developed was compared with LAMP, and the primers used in the real-time LAMP and LAMP were listed in 2O was used as a negative control. The primers used in the real-time LAMP and LAMP were listed in The EHP DNA was used as the template, and ddH2O as a control template to test the set of primers. The S type curve demonstrated a positive result, while the smooth straight line a negative result. From the appearance of the amplification curves, the set of primers demonstrated increased fluorescent signals without any amplification peak in the negative control  1. (B) Amplification curve of EHP 2. (C) Amplification curve of EHP 3. (D) Amplification curve of EHP 4. (E) Amplification curve of the negative control.The ΔRn vs. cycle graph was plotted automatically by StepOne Real-Time PCR System. ΔRn is the fluorescence unit minus the baseline, the graph depicts the ΔRn on the Y axis and cycle number on the X axis. (A) Amplification curve of Click here for additional data file.10.7717/peerj.5993/supp-2Supplemental Information 2Enterocytozoon hepatopenaei (EHP). (A) Amplification curve of EHP 3. (B) Amplification curve of EHP 4. (C) Amplification curve of white spot syndrome virus (WSSV). (D) Amplification curve of macrobrachium rosenbergii noda virus (MrNV). (E) Amplification curve of infectious hypodermol and hematopoietic necrosis virus (IHHN). (F) Amplification curve of the negative control.Three different pathogen templates were performed along with Click here for additional data file.10.7717/peerj.5993/supp-3Supplemental Information 30 times to 10−6 times for Enterocytozoon hepatopenaei (EHP) positive sample DNA. (NG) Negative control.The fluorescence unit vs. cycle graph was plotted from the data of the StepOne Real-Time PCR System. Serial 10-fold dilutions ranging from 10Click here for additional data file.10.7717/peerj.5993/supp-4Supplemental Information 4Enterocytozoon hepatopenaei (EHP). (D-I) Corresponding to serial 10-fold dilutions ranging from 100 times to 10−6 times for EHP positive sample DNA, respectively. (J) Negative control.(A) Negative control. (B) Positive control. (C) Click here for additional data file.10.7717/peerj.5993/supp-5Supplemental Information 5(A) Amplification curve of real-time Loop-Mediated Isothermal Amplification. (B) Amplification curve of conventional Loop-Mediated Isothermal Amplification. (C) Amplification curve of the negative control.Click here for additional data file.10.7717/peerj.5993/supp-6Supplemental Information 6Clinical samples testing was carried out using the established real-time LAMP method. (A) Positive control (B) Negative control (C-P) Clinical samples 1–14.Click here for additional data file.


Natural dissolved organic matter (DOM) comprises a broad range of dissolved organic molecules in aquatic systems and is among the most complex molecular mixtures known. Here we show, by comparing detailed structural fingerprints of individual molecular formulae in DOM from a set of four marine and one freshwater environments, that a major component of DOM is molecularly indistinguishable in these diverse samples. Molecular conformity was not only apparent by the co-occurrence of thousands of identical molecular formulae, but also by identical structural features of those isomers that collectively represent a molecular formula. The presence of a large pool of compounds with identical structural features in DOM is likely the result of a cascade of degradation processes or common synthetic pathways that ultimately lead to the formation of a universal background, regardless of origin and history of the organic material. This novel insight impacts our understanding of long-term turnover of DOM as the underlying mechanisms are possibly universal. Dissolved organic matter (DOM) in aquatic systems is among the most complex molecular mixtures known. Here the authors show that a major component in DOM is molecularly indistinguishable in marine and freshwater environments, which could reflect universal mechanisms behind long-term DOM turnover. Mostly because of this molecular complexity, full structural elucidation of DOM has been impossible to date. The structures of only a few percent of the many hundreds of thousands of the individual compounds in DOM are known5. The remarkable advance of ultrahigh-resolution analytical techniques, namely Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) and high-field nuclear magnetic resonance spectroscopy (NMR), has allowed fundamentally novel insights into the molecular composition of DOM. More than 10,000 molecular formulae and hundreds of structural features have been identified with their help to date6. The molecular structures behind these molecular formulae, and the spatial arrangement of the identified structural features, however, are still largely unknown and remain an ongoing analytical challenge7.DOM comprises a complex molecular mixture8 and, consequently, on its susceptibility to microbial and abiotic transformations9. For instance, DOM in freshwater is often enriched in lignin-derived polyphenols that originate from vascular plant debris11. The abundance of these polyphenols renders the waters brown in color as typical for many rivers, lakes or wetlands. Marine DOM is comparably poor in these compounds12. Aside from the obvious source-specific molecular properties of DOM, on which a rich body of literature exists, there is a component in DOM that is molecularly indistinguishable across all natural aquatic systems16. Evidence for this universal component in DOM was found with help of ultrahigh-resolution FT-ICR-MS and multidimensional nuclear magnetic resonance (NMR). Thousands of molecular formulae found by FT-ICR-MS in DOM in the deep ocean are also present in rivers, lakes, wetlands and even degraded plant leachate and microbial cultures18. A common structural feature, as indicated by a combination of NMR and FT-ICR-MS, are carboxylic-rich alicyclic moieties (CRAM)15 and material derived from linear terpenoids (MDLT)16. It was hypothesized that long-term degradation processes such as a cascade of photo-degradation together with microbial mediated transformation of DOM leads source-independently to these common structural features20. This molecularly indistinguishable fraction of DOM may withstand decomposition over millennia and remain in solution during transport from the site of production at the sea surface or on the continents into the deep sea21. However, a largely unexplored level of molecular diversity exists for DOM because each molecular formula is represented by a range of different isomers, i.e., compounds with the same molecular formula but different molecular structures23. Similarly, there is a high degree of freedom to arrange the structural features identified in DOM via NMR. Possibly, the molecular similarity of the universal DOM background may simply be the reflection of our inability to fully characterize DOM on a structural level. This lack of knowledge is of concern because it prevents a truly mechanistic understanding of organic matter cycling in freshwater and marine systems.The original source exhibits a strong influence on DOM molecular compositionHere we took the analytical challenge to assess the molecular diversity of DOM beyond the molecular formula level by performing extensive experiments on individual molecular formulae in an ultrahigh-resolution FT-ICR-MS. This was done to test the hypothesis that DOM in the various aquatic environments and from the various sources only apparently shares identical molecular features, and that differences emerge on an isomeric level. The results demonstrate the opposite and identical structural features are identified for the isomers that represent a molecular formula across all tested environments. This indicates that the underling mechanism of DOM turnover is universal for this component of DOM.24, but fragmentation experiments uncovered different molecular structures behind the same molecular formulae in such experiments25. Exemplary analysis of benzenetricarboxylic acid isomers with identical molecular formulae as model compounds illustrated that even slight structural differences lead to sharply different fragmentation patterns in FT-ICR-MS  for fragmentation experiments from full range mass spectra of the five DOM samples  from decarboxylation and water (H2O) as a result of alcohol dehydration or formation of anhydrides30  and methanol (CH3OH). In addition, very minor fragmentation signals in terrestrially influenced samples likely originated from polyphenolic compounds, a compositional feature indicative of the presence of vascular plant debris. Despite a clear terrigenous origin, fragmentation of functional groups that are typical for lignin (methoxy groups)31 were not observed. This is likely because these compounds are readily degraded33 and were already removed on their journey from soils to the lake or into the coastal ocean. The qualitative evaluation of fragmentation patterns, i.e., the type of occurring neutral losses, agrees with earlier fragmentation studies on DOM30.We chose six individual nominal masses  is here defined as the FT-ICR-MS signal intensity of the fragment ion (IF) divided by the sum intensity of all major signals (ITot), which includes the precursor ion and fragments attributed to that precursor. In a comparison of relative fragment ion intensities, a slope of 1 is expected if two samples contain identical compounds. We found highly significant positive correlations  between the fragment intensities of all samples and only very minor and non-significant deviations from a slope of one, with Δsl ranging from 0.008 (NADW/EMW) to up to 0.243 (Lake/AABW)  based on FT-ICR-MS signal intensities to describe the molecular differences between the DOM samples on the molecular formula level and on the structural level via fragmentation. The analysis was performed first for all detected, unfragmented molecular formulae (6166) in the full-range, unfragmented mass spectra Fig. . The higThe high similarity of relative fragment ion intensities is remarkable, considering the different origins of the samples. Regardless of origin, compounds that shared the same molecular formula in vastly different aquatic environments also shared indistinguishable structural features, even when assessed by the most advanced analytical techniques. This similarity did not only apply to the type of functional groups, but most likely also to their position in the given molecule. Our experiments with model compounds  over degradation and mixing, and a decreasing occurrence of site-specific DOM compounds. It has recently been shown that time is a driver over DOM molecular composition and diversity across aquatic ecosystems37. In theory, it is also possible that the 54 fragmented formulae share a common set of source-material in the different environments, e.g., specific biomolecules from terrestrial organic matter input. However, we chose the compounds across the entire spectrum of DOM compounds and not from a subgroup with specific molecular composition  that is inherent of a specific ecosystem is termed molecular α-diversity. It is important to note that as in ecology, α-diversity was determined in our study by a random sample. The comparison of α-diversity between different samples is only possible if the size of the samples is the same, since the number of species (or molecules), and thus the diversity, usually increases with increasing sample size. We accounted for these conceptual considerations by analyzing all samples at the same DOC concentration and collected the same number of FT-ICR-MS scans for each sample.14. Degradation processes could be abiotic  or biotic, and particularly microbial reworking is an important process in this context. Marine microbes remove DOM from the water, but they also produce new DOM. In fact, the DOM released by marine microbes is more diverse than the initial substrates and, thus, they contribute to molecular diversification25. Part of the source-specific DOM component, however, seems to be resistant towards photo- and year-long biodegradation40, and long timescales are necessary for the observed prevalence of the universal DOM component in the deep sea. Previous studies were stating the same hypothesis, that common processes may form a large pool of similar DOM structures across different environments39. Our structural comparison of DOM from different marine and freshwater environments and our estimates of isomeric diversity are confirmative of these ideas presented previously39. The question arises whether the same holds true for N and S containing compounds. We did not exclude these compounds in our study, however, only few of the fragmented formulae contain these heteroelements as the original unfragmented signals are usually not as abundant as those with formulae containing only C, H, and O. We would like to acknowledge that even though the new data presented cannot conclusively settle the discussion on DOM molecular diversity, it represents a major step towards an improved understanding of the structure of the DOM pool.Cascades of degradation processes and mixing in aquatic environments likely increase molecular α-diversity through the stepwise formation of a complex mixture with a high number of isomers behind each molecular formulaOur study represents the first comparison of comprehensive DOM molecular structural features in different aquatic environments, including the deep sea and a eutrophic peat lake. Within our analytical window, we found a component in DOM that is molecularly indistinguishable across the globe. This novel insight impacts our understanding of long-term turnover of DOM as the underlying mechanisms are possibly universal, at least for this component. Affinity of consumers towards common biomolecules is one of the commonalities of degradation processes in all aquatic environments, but the structural similarity of DOM seems to go far beyond what can be explained by this process.41 but of different origin as NADW is formed in the North Atlantic and AABW in the Southern Ocean. Highly saline EMW from the outflow of the Mediterranean Sea is younger than oceanic deep-water masses but also characterized by a refractory DOM signal42. A sample from the North Sea, a continental shelf sea in Europe that contains a terrigenous component and freshly produced DOM43, was taken with a bucket from the sea surface. Water from a eutrophic peat lake was collected in January 2011 in Meyerhausen, Germany, and is presumably dominated by terrestrial vascular plant decomposition products44.Samples were collected from five different sites Fig. . Exact p45 using a Shimadzu (Japan) TOC-VCPH/CPN Total Organic Carbon Analyzer equipped with ASI-V auto sampler. We controlled the accuracy of the measurement for every run by analyzing deep-sea reference samples provided by the University of Miami (Dennis Hansell). The error of DOC was below 2.8 µmol C L-1.Water was filtered through 0.7 µm GF/F glass microfiber filters , acidified with HCl  to pH 2 and stored at 4°C in the dark. Analysis of DOC concentrations was performed via high-temperature catalytic oxidation method46. Procedural blanks were prepared by processing ultrapure water the same way as DOM samples. DOC concentrations in the resulting extracts were below the detection limit. We performed mass spectrometric analysis of DOM extracts via FT-ICR-MS on a solariX Fourier transform ion cyclotron resonance mass spectrometer with 15 Tesla magnet . The system was equipped with an electrospray ionization source  applied in negative ionization mode. Methanol extracts were mixed with ultrapure water (50:50 v/v) for FT-ICR-MS analysis and diluted to a final DOC concentration of 20 mg C L−1. For each measurement we accumulated 500 scans in the mass window of 150–2000 Da. Including test runs and the analysis of NEqPIW deep-sea reference, we needed more than 600 h of instrument time to acquire all full-range, isolation and fragmentation mass spectra. We calibrated spectra internally with a reference mass list using Data Analysis software Version 4.0 SP4. The mass error of the calibration was <0.06 ppm for all samples. We used MatLab routines developed by our working group for molecular formula assignment and further data processing. Only peaks with a signal-to-noise ratio of S/N = 5 or higher that fulfilled the criteria by Koch et al.47 and Rossel et al.13 were considered including the elements C, H, O, N, S, and P with respect to the following criteria C0–60, H0–∞, N0–4, S0–2, P0–1, O/Cmax = 1, and H/Cmin = 0.3. All molecules were detected as singly charged ions. To test the reproducibility and stability of the FT-ICR-MS analysis, SPE-DOM from North Equatorial Pacific Intermediate Water (NEqPIW) was analyzed with the same settings twice a day48. Finally, we normalized peak intensities of the peaks with assigned molecular formula to the sum of peak intensities for multivariate statistical analysis. A total of 9794 resolved masses of singly charged, intact compounds were detected via FT-ICR-MS. Thereof, 6166 masses could be assigned to molecular formulae. Most of the remaining detected masses (96%) could be assigned to a molecular formula by including 13C1 and 15N1 into the list of considered elements and can therefore be related to isotopologues . Molecular categories according to criteria modified after Šantl-Temkiv et al.49 were calculated for all detected molecular formulae largely based on the modified aromaticity index AImod50. The following groups were differentiated: combustion-derived polycyclic aromates , oxygen rich polyphenols , oxygen poor polyphenols , highly unsaturated compounds , and aliphatic compounds   were extracted via solid phase extraction using a commercially available PPL sorbent (Agilent)2 homologous series. Thus, they are representative for the molecular diversity of DOM. The mass range around 370 Da was chosen because the high signal intensities enable acquisition of the most detailed structural fingerprints. Fragmentation of DOM samples was carried out with methanol extracts that were mixed with ultrapure water (50:50 v/v) to give a final concentration of 100 mg C L−1. For each spectrum 150 scans were acquired with a data acquisition size of 4 Mword. The collision voltage was 15.0 V and the detection range was set from m/z 125–2000. Collision induced fragmentation in the quadrupole with subsequent detection via FT-ICR-MS resulted in a total of 1953 distinct molecular fragments, of which 265 derived from the 54 molecular formulae that were common in all samples.For fragmentation, ions were first isolated in 0.4 Da windows in a quadrupole unit of the FT-ICR-MS. Ions were then transferred into a hexapole unit, where they accumulated for 2 s and collided with neutral argon gas. The molecular formulae that were chosen for fragmentation represent all the major molecular categories  from the masses of the ions detected on the respective isolated nominal mass  and the precursor ion peak intensity. Based on these relative peak intensities, a linear regression model was calculated . Therefore, we included only the most intense fragment ions for the statistical analysis, which were resulting from neutral losses of mainly CO52.DOC and sample metadata are included in this published article and its Supplementary Information files. Mass spectrometric data has been deposited in the PANGAEA Open Access library (10.1594/PANGAEA.888852)Supplementary Information


Kaposi sarcoma (KS) is a mesenchymal tumour caused by KS-associated herpesvirus and is an AIDS-defining illness. Despite a decline in incidence since the introduction of combination anti-retroviral therapy, KS remains the most common cancer in people living with HIV in sub-Saharan Africa, where it causes significant morbidity and mortality. This review reflects on recent epidemiological data as well as current management, unmet needs and future perspectives in the treatment of HIV-associated KS with particular emphasis on the potential role of immune checkpoint inhibitors. Kaposi sarcoma (KS) is an angio-proliferative disorder that was first described in its classic form by the Hungarian physician Moritz Kaposi in 1872. It is categorised in different disease types that are all aetiologically associated with KS-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8). The malignant cell of KS is the spindle cell which is infected by KSHV and is believed to be of lymphatic endothelial origin. KS lesions are composed of a combination of abnormal vascularity, inflammation and fibrosis with hemosiderin deposits.1; allograft transplantation-associated (iatrogenic) KS; and AIDS-associated (epidemic) KS, which is characterised by potentially aggressive clinical features.There are four widely recognised epidemiological forms of KS: classic KS, which is a relatively indolent disease that mainly affects elderly men from the Mediterranean basin and Middle East; African (endemic) KS, which pre-existed the HIV/AIDS pandemic3. WHO-GLOBOCAN data indicate that there were 41,799 new KS diagnoses  and 19,902  KS deaths globally in 20184.Despite being a relatively infrequent malignancy worldwide, KS remains the most common cancer in people living with HIV (PLWH) in the African continent and is one of the most common tumours, especially in children in central, eastern and southern Africa5 and to a significant improvement in KS prognosis7. However, KS still accounts for significant morbidity and mortality, particularly in sub-Saharan countries. Although the incidence of KS fell with the cART-induced rise in CD4 T-cell count and immune reconstitution, the risk of developing KS in PLWH with a normal CD4 T-cell count remains substantially higher than in the general population8.The introduction of combination anti-retroviral therapy (cART) led to a remarkable decline in AIDS-KS incidence10, suggesting a proportion of cases to be fully sensitive to cART-mediated immune reconstitution. After starting cART, patients with anti-retroviral–naïve HIV recover their immunity, at least in part, within months. Responses to cART alone are variable. Complete remission of KS occurs in 20 to 80% of patients and is more frequent in cART-naïve patients with limited-stage disease who are compliant with HIV therapy11. The mechanism of KS regression is thought to be immunological and specifically associated with reconstitution of an effective cytotoxic cell–mediated response targeting KSHV. As KS can rapidly progress and affect vital organs, patients with advanced-stage KS . Treatment responses were often non-sustained in these initial trials that predated the introduction of cART. In advanced-stage KS, the concomitant use of cART and liposomal anthracyclines achieves a 70% overall response rate with prolonged remission19. Over time, improvements in the understanding of kinetics of immune reconstitution and the consolidating knowledge that cART is an effective and sufficient therapy for people with early stage KS (T0)20, but not for patients with advanced disease (T1 stage), led to the consolidation of a stage-stratified approach in patients with KS. This approach, proposed in the British HIV Association guidelines for HIV malignancies (BHIVA)21, supports the use of cART alone for early-stage KS (T0), reserving the addition of liposomal anthracycline chemotherapy for more aggressive disease (T1), based on evidence of efficacy in PLWH with visceral KS. The effectiveness of a stage-stratified approach to KS was further consolidated by a landmark study that described a cohort of 469 patients, confirming that this approach achieves high survival rates in patients with advanced KS  and reduces exposure to chemotherapy in patients with early-stage disease (5-year OS 95%)22.Prior to the advent of cART, a number of agents, including interferon-alpha, thalidomide, retinoids and cytotoxics such as vincristine and bleomycin, were used in the treatment of HIV-KS. However, the advent of liposomal anthracyclines in the early 1990s is recognised as a significant advancement in the chemotherapy of KS and has become the gold standard of care. Their role has been consolidated by level-I evidence from three randomised controlled trials that showed better response rates and favourable toxicity profiles3)25. The immunopathology of cART-refractory KS is complex. Mechanistic studies have recognised that this type of disease is associated with T cell–immune senescence as a key pathophysiologic trait and that in some patients it has an indolent clinical course similar to that of the classic subtype26. In our HIV-cancer centre, this group of patients usually receives multiple cycles of chemotherapy alternating between liposomal anthracycline and paclitaxel. Sequential alternation of non–cross-reacting chemotherapy agents has been found to be an effective approach to control HIV-KS in a series of 20 patients however, no comparative studies are available and this treatment strategy may lead to increased chemotherapy toxicities27. Cardiotoxicity is linked to cumulative dosage of anthracyclines28, although the risk is lower with the liposomal formulation and high cumulative dosages of taxanes, used as a second-line therapy, are associated with irreversible peripheral neuropathy29.Even with the notable advancements in the systemic management of KS, up to 15% of patients develop progressive disease despite achieving good immune-virologic control of HIV with undetectable plasma HIV-1 RNA and adequate CD4 T-cell counts .31. This is one of the main unmet needs in the management of KS in resource-poor countries and research and development as well as health policy efforts should therefore focus on the availability of oral, easily delivered, and inexpensive regimens in these regions.Although recent efforts have made cART available in most resource-limited settings, access to chemotherapy remains a major problem. More affordable chemotherapy agents such as  doxorubicin, bleomycin, vincristine and etoposide may be currently administered in sub-Saharan Africa but they are associated with increased toxicity and lower efficacy for HIV-associated KS. Moreover, the infrastructure and expertise necessary to safely reconstitute and deliver intravenous chemotherapy are not available in many hospitals in Africa32.A recent study conducted by the AIDS Clinical Trials Group and the AIDS Malignancy Consortium in Africa and Brazil demonstrated that oral etoposide is inferior to both intravenous paclitaxel and intravenous combination bleomycin and vincristine in advanced disease and that paclitaxel is somewhat better than combination bleomycin and vincristine+ and CD8+ T-cell co-stimulation. Notably, it is logically presumed with limited supportingin vivo data that the latter immunological factors are critical drivers in the development of KS33. In a phase 2 study, 22 patients who received treatment achieved an overall response rate of 73%. Responses were rapid and occurred in HIV-positive and HIV-negative patients with advanced KS and in heavily pre-treated subjects34. A trial conducted with a similar immunomodulatory agent, lenalidomide, had somewhat less favourable results with a response rate of 40% using the AIDS Clinical Trial Group (ACTG)-defined criteria and a 0% Physical Global Assessment (PGA) criteria response rate35 at week 48. Uldricket al.36 published phase 2 trial results of the humanised monoclonal antibody bevacizumab, which targets VEGF-A, in HIV-associated KS. The overall response rate was only 31% despite the interesting role of VEGF-A in KS pathogenesis as a paracrine and autocrine growth factor for KS cellsin vitro37. This points to the need forin vivo experimental data to support a definitive role for VEGF-A in the development of KS. Disappointing results have also been obtained with matrix metalloproteinase inhibitors38 and other angiogenesis inhibitors39. Given that activation of platelet-derived growth factor (PDGF) and tyrosine kinase (TK) receptor c-kit has been demonstrated to have a role in KS pathogenesis bothin vitro andin vivo, imatinib, a selective inhibitor of the TK Abl and c-kit, has been tested in HIV-KS with promising results40. Similarly, KSHV-encoded proteins upregulate the mitogen-activated protein kinase (MAPK) pathway41, and a clinical trial of the use of selumetinib, an inhibitor of this pathway, in HIV-KS is being completed. Rapamycin has been shown to be effective in transplantation-associated KS and has also been found to have activity in AIDS-KS but has significant drug–drug interactions with cART. Despite the pharmacokinetic interactions, that result in more than 200-fold differences in cumulative rapamycin doses between participants on protease inhibitor (PI)- and non-nucleoside reverse transcriptase inhibitor (NNRTI)-containing regimens, treatment was well tolerated42.It must be noted that there is no accepted animal model for KS or KSHV infection for testing of drug therapies prior to clinical trials. Hence, in recent years, multiple targeted therapies for KS, especially oral medicines, have been directly routed to human trials. The most promising results have been achieved with pomalidomide, an orally available derivative of thalidomide that is less neurotoxic and has immune-modulatory, anti-angiogenic, and anti-proliferative activities. The effects against KS include modulation of tumour necrosis factor-alpha, interleukin-6, and vascular epithelial growth factor (VEGF) and enhancement of CD4de novo development of HIV-associated KS in virally suppressed patients with a preserved CD4 T-cell count remain unclear. Anti-cancer immune response is closely linked to adaptive T-cell responses as part of a “cancer-immunity cycle”43. This immune response is initiated with antigen priming of T lymphocytes: antigens are released by dying tumour cells and presented by dendritic cells via the major histocompatibility complex to the T-cell receptor. Primed T cells then traffic from the lymph node to the tumour site where they recognise and kill the cancer cells in the effector phase of adaptive immune response. Tumours have developed immune tolerogenic strategies that are delivered through complex mechanisms, including the modulation of multiple co-inhibitory and co-stimulatory pathways. Two immune checkpoints—cytotoxic T-lymphocyte–associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) pathways—respectively inhibit the priming and effector phase of cancer-specific immune response. They are therefore of great clinical interest in the development of new cancer immunotherapies.The mechanisms behind the persistence or44.In vitro studies indicate that PD-L1 blockade reverses immune dysfunction of HIV-specific CD8 T cells with increased survival proliferation and cytokine production45. Hostet al. have also demonstrated that KSHV infection of primary human monocytes upregulates PD-L1 transcription and expression which highlight a new immune evasion strategy for KSHV46.In the context of chronic viral infections, including HIV and malignancy, the upregulation of PD-1 on cytotoxic T lymphocytes is a well-described mechanism of immune tolerance. In the HIV infection setting, chronic inflammation can lead to the exacerbation of immune exhaustion pathways with a subsequent decline in cancer immune surveillance and the development of a favourable oncogenic microenvironmentin vivo, PD-L1–positive KS was associated with greater T-cell and macrophage infiltrate, hallmarks of an exhausted immune response to cancer47.In cART-refractory KS, our group has evaluated the potential for the PD-1 immune tolerance pathway as a mechanism underlying the pathogenesis and development of active KS in patients with well-controlled HIV stable on cART. Though limited by a small sample size of 10 patients, we confirmed PD-L1 upregulation with evidence of weak cytoplasmic PD-L1 expression in 50% of cases. Though differing from the intense membranous pattern of expression in epithelial malignancies that respond to PD-L1/PD-1 blockadeet al. reported on the efficacy and toxicity of immune checkpoint inhibitors (anti-PD-1 nivolumab and pembrolizumab) in nine patients with HIV-associated KS48. The authors conducted a retrospective review of a heterogeneous group of patients: four patients had cutaneous disease only (T0), five patients had visceral disease (T1), seven out of nine patients had well-controlled HIV, and two had detectable plasma HIV RNA. Five patients had a good-risk immune status (I0) with a CD4 T-cell count above 200 cells/mL, and four patients had a CD4 count below the 200 cells/mL threshold48. The patients were also heterogeneous in terms of prior treatment, having received from none up to four lines of systemic therapy prior to anti-PD-1 blockade, including liposomal anthracycline, paclitaxel, lenalidomide and bortezomib. One patient achieved complete remission, five out of nine patients achieved partial response, and three patients had stable disease. No one experienced KS progression and they were still on treatment at the time of reporting. Patients were evaluated for response every 4 weeks with KS response criteria defined by the AIDS Malignancy Consortium. The median progression-free survival has not been reached in the nine patients at a median follow-up of 5 months. No serious toxicity above grade 2 was observed in the study, and the most common side effects were fatigue, muscle aches and pruritus. Similar results were published in two cases of HIV-negative KS treated with nivolumab49. The authors’ publication supports our provocative findings that PD-1 checkpoint blockade could have a therapeutic role in HIV-KS even in the presence of weak or no PD-L1 tumour expression in a similar way that good responses are observed in other virally induced tumours such as polyomavirus (MCPyV)-associated Merkel cell carcinoma50. Nevertheless, the study is preliminary and does not lead to definitive conclusions as to the positioning of PD-1 therapy in KS. Longer prospective trials with the aim to better evaluate this treatment strategy are under way51.Since our study, Galanina54 and limited but the effect of immune checkpoint inhibitors in this setting remains an area of interest where prospective studies are warranted. Especially because immune checkpoint blockade is associated with a risk of life-threatening autoimmune pathologies55, studies of PD-1 blockade as a curative strategy in HIV should proceed with caution. Recent data are also highlighting the role of the PD-1 pathway in the priming of HIV T-cell responses; therefore, extra consideration should be taken in the timing of blocking this signalling pathway in order to avoid potential negative effects on the induction ofde novo T-cell responses56. Benefits may be limited to specific groups of patients with currently undefined predictors of response. Given the favourable toxicity profile of PD-1 blockade in the study by Galaninaet al., the PD1 blockade described is more justified when the HIV research is combined with a cancer treatment strategy such as in KS.An additional benefit to the anti-tumour effect of anti-PD-1 therapy might be the potential role in HIV cure strategies. This immunotherapy is potentially able to reactivate latent HIV-1 and enhance HIV-specific antiviral response. The results on depleting latent HIV reservoirs so far are inconsistentThe introduction of cART has not resolved the challenge of HIV-associated KS. Unmet needs include access to liposomal anthracycline chemotherapy in sub-Saharan Africa for patients with advanced disease and the development of new treatment strategies for patients with cART-refractory KS who are currently intermittently treated long term with potentially toxic chemotherapy. The immune checkpoint inhibitor certainly represents a very interesting area to explore further given the parallel potential as an HIV cure strategy.