Cofactor insertion is an intricately controlled process, and the large subunit of almost all Hyd enzymes has a C-terminal oligopeptide extension that is endoproteolytically removed as the final maturation step. This extension might serve either as one of the recognition motifs for the endoprotease, as well as an interaction platform for the maturation proteins, or it could have a structural role to ensure the active site cavity remains open until the cofactor is inserted. To distinguish between these alternatives, we exchanged the complete C-terminal extension of the precursor of Escherichia coli hydrogenase 2 (Hyd-2) for the C-terminal extension of the Hyd-1 enzyme. Using in-gel activity staining, we demonstrate clearly that this large subunit precursor retains its specificity for the HybG maturation chaperone, as well as for the pro-HybC-specific endoprotease HybD, despite the C-terminal exchange. Bacterial two-hybrid studies confirmed interaction between HybD and the pro-HybC variant carrying the exchanged C-terminus. Limited proteolysis studies of purified precursor and mature HybC protein revealed that, in contrast to the precursor, the mature protein was protected against trypsin attack, signifying a major conformational change in the protein. Together, our results support a model whereby the function of the C-terminal extension during subunit maturation is structural.The active site of all [NiFe]-hydrogenases (Hyd) has a bimetallic NiFe(CN) A still unresolved issue on the maturation pathway of the catalytic, or large, subunit of [NiFe]-hydrogenases (Hyd) is what role the C-terminal oligopeptide extension plays during the maturation process: Does it act as a recognition motif allowing interaction with maturation proteins, or does it function as a form of “intra-molecular” chaperone that constrains the large subunit precursor in a conformation allowing access of the maturation machinery, which then interacts elsewhere on the protein? Or does it do both?With the exception of the large subunit of sensory and Ech hydrogenases , the fin2CO moiety of the cofactor is the first component to be delivered to the precursor, probably by the HypC protein. In bacteria synthesizing more than one Hyd, paralogues of HypC exist, e.g., HybG in Escherichia coli, and these paralogues deliver the Fe(CN)2CO moiety to a specific Hyd enzyme . The templates used were the plasmids pASK-hybC (hyaB (hyaB plasmid (hybC in pCAN-hybC (proc (see Amino acid exchanges of the A550S and V551 T (numbers are based on untagged protein) were introduced simultaneously in ASK-hybC  and pASKbC  using the appropriate combination of oligonucleotides . A column containing 1 ml bed-volume of resin was pre-equilibrated with 10 ml of anaerobic buffer A  followed by application of filtered , anaerobically prepared crude extract  containing the His-tagged protein at a flow rate of 1 ml min−1. Unbound proteins were washed from the column with 10–15 column volumes of buffer A, followed by a washing step (3 column volumes) using buffer A containing 30 mM imidazole. Bound, His-tagged proteins were eluted with buffer A containing 300 mM imidazole. After elution, pooled eluate containing the purified protein(s) of interest was buffer-exchanged into buffer C  using a Sephadex PD-10 column .N-terminally His-tagged HybC variants were purified after overproduction in strain CB17  by metalproc  in a 100 μl reaction volume of 200 mM HEPES pH 7.8 containing 200 mM NaCl and 40 mM CaCl2 were incubated with 1 μg of trypsin or chymotrypsin and 10 μl aliquots were removed at the indicated times and immediately added to boiling SDS buffer to terminate the reaction. Polypeptides were separated in denaturing SDS-PAGE (Aliquots (100 μg) of pro-HybC or HybCSDS-PAGE  containiC-terminally Strep-tagged HypC and HybG were purified as described in These experiments were performed as described by 2CO moieties of the cofactor. Current evidence strongly suggests the latter role  carrying the Hyd-1-specific ST instead of the Hyd-2-specific AV amino acid residues at these positions , hypC (CB19), hyaD (CB16), or hybD (CB17). After anaerobic growth in minimal medium with glucose as a carbon source, cell-free extracts were prepared, and the Hyd-2 enzyme activity profile of each strain was analyzed after separation of enzyme complexes in native-PAGE .The C-terminal peptide on pro-HybC, the large subunit precursor of Hyd-2, functions either as a recognition motif for the Hyp cofactor-insertion machinery or it maintains the empty active site in an open conformation, facilitating insertion of the nickel and Fe(CN)ter role . To demoter role , for theter role . A variater role . This co role of Hyd-2 . Pro-Hya or HypC . To deteant CB18 . No cleaG mutant . When thG mutant ,C. This Nof Hyd-2E. coli, HyaD is the maturation endoprotease that is specific for pro-HyaB, while HybD is specific for pro-HybC (hybD (hyaD (§) nor introduction of the AV → ST exchanges (pro-HybCST) affected the protease specificity of the precursor; all four derivatives retained an absolute dependence on HybD . Moreover, the results confirmed that this pro-HybC§ST variant generated a lower Hyd-2 enzyme activity compared with pro-HybCST. In order to demonstrate the protease specificity for HybD of all four pro-HybC derivatives, we performed bacterial two-hybrid analysis, whereby peptide T25 of adenylate cyclase was fused to the N-terminus of the HybC derivatives, while the protease HybD was fused with peptide T18 of adenylate cyclase . This result indicates that the interaction between HybD and the HybC precursors was highly specific.In contrast to what was observed for fusions with pro-HybC, a fusion of the Hyd-1 large subunit precursor, pro-HyaB, or of pro-HyaBproc, the processed form of HybC lacking the 15-amino-acid long C-terminal peptide, have different conformations .HybG is required for the delivery of the Fe(CN)pro-HybC  and HybGpro-HybC . Our finpro-HybC  and recepro-HybC . To provpro-HybC . As a copro-HybC  and indepro-HybC . Full-lepro-HybC , suggestE. coli, and which are part of the conserved D-P-C-X-A-C-X-X-H motif that forms the C-terminus of most nickel hydrogenase large subunits, are interchangeable and not essential for either HybG or endoprotease specificity; however, the exchange from AV to ST in pro-HybC did impair the efficiency of maturation. It is likely that this effect is due to the proximity of these residues to the crucial fourth cofactor-coordinating cysteinyl residue (Cys-4), which ultimately bridges the two metals of the NiFe-cofactor in the active site, thus conferring activity upon the cofactor (T. kodakarensis revealed that a β-strand formed within the C-terminal extension and interacted with two β-strands in the N-terminus of the polypeptide to form a β-sheet structure. This conformational change displaced the Cys-4 residue away from the other three coordinating Cys residues (2CO and nickel moieties to the precursor.Our results support the conclusion that the C-terminal oligopeptide extension on the large subunit of Hyd-2 has a chaperone-like function  and do ncofactor . The impresidues , consequproc became significantly more resistant to trypsin treatment when compared with pro-HybC. Moreover, interaction with HybG also protected the “open” conformation of the protein against trypsin, which corroborates our proposal that HybG possibly interacts with the N-terminal region of the protein, as has been shown for the nickel delivery protein HypA during its interaction with HyhL (proc (§ST retained its specificity for HybD.After endoproteolytic cleavage, the C-terminus is suggested to “flip”back forming a short α-helix closing the active site, thus allowing Cys-4 to bridge the two metals. This hypothesis is supported by the recently resolved structure of the mature HybC protein . Our limith HyhL . MoreovehL (proc . ContrarhL (proc , the resThe datasets generated for this study are available on request to the corresponding author.CP, CT, and KN designed and performed the experiments and analyzed the data. CP and RS conceived the study, interpreted the data, and drafted the manuscript. All authors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The aim of the present study is to check whether we can replicate, in an independent series, previous results showing that the molecular study of pituitary-specific gene expression complements the inmunohistochemical identification of pituitary neuroendocrine tumours. We selected 112 patients (51 (46.4%) women; mean age 51.4±16 years; 102 macroadenomas (91.9%), 9 microadenomas (8.1%)) with complete clinical, radiological, immunohistochemical and molecular data from our data set of pituitary neuroendocrine tumours. Patients were different from those previously studied. We measured the expression of the pituitary-specific hormone genes and type 1 corticotrophin-releasing hormone and arginine vasopressin 1b receptors, by quantitative real-time polymerase chain reaction using TaqMan probes. Afterwards, we identified the different pituitary neuroendocrine tumour subtypes following the 2017 World Health Organization classification of pituitary tumours, calculating the concordance between their molecular and immuhistochemical identification. The concordance between molecular and immunohistochemical identification of functioning pituitary neuroendocrine tumours with the clinical diagnosis was globally similar to the previous series, where the SYBR Green technique was used instead of TaqMan probes. Our results also corroborated the poor correlation between molecular and immunohistochemical detection of the silent pituitary neuroendocrine tumour variants. This discrepancy was more remarkable in lactotroph, null-cell and plurihormonal pituitary neuroendocrine tumours. In conclusion, this study validates the results previously published by our group, highlighting a complementary role for the molecular study of the pituitary-specific hormone genes in the typification of pituitary neuroendocrine tumours subtypes. In 2017, the World Health Organization (WHO) classification of tumours of the pituitary gland superseded its previous 2004 version,6].,[6].POMC.,[6].POMGT),,,, we, we,[,[,GH gene in functioning ST PitNET depending on the treatment with somatostatin analogues, we used the Mann Whitney test. We considered P values of less than 0.05 to be statistically significant. Statistical analysis was performed with SPSS 24.0 software (IBM Software).Qualitative variables, including PitNET subtypes, were expressed as absolute and relative frequencies. Participant age was expressed as mean ± standard deviation (SD). Molecular variables showed a non-normal distribution (Kolmogorov-Smirnov test), so we chose percentiles as the measure of distributions and reported them as median plus interquartile range (IQR). We calculated the Cohen’s kappa coefficient to measure concordance between the clinical, IHC and molecular identification of the different subtypes of PitNET (κ = 1 representing complete concordance and κ ≤ 0 null concordance). To determine the relationship between the administered doses of cabergoline and the expression of the PRL gene in FLT, we used Spearman’s correlation. In order to determine differences in the expression of The series studied in the present work was composed of 112 tumours, 48 (42.8%) of which were clinically functioning. The demographic and clinical characteristics of the patients are shown in Following the 2017 WHO system, we classified the different subtypes of PitNET according to the dominant pituitary-specific hormone gene or protein expression in the IHC and molecular studies . In S4 TOnce we identified the different subtypes of PitNET by IHC and molecular study, the clinical and biochemical information allowed us to define the functioning and silent variants of the tumours. Consequently, we show the classification and prevalence of the different PitNET subtypes according to their clinical, molecular and IHC identification .et al.[Our results replicate those obtained by Sanchez-Tejada et al.. Both stRemarkably, the concordance of the molecular analysis with the clinical diagnosis was complete or almost complete for Cushing disease and functioning thyrotropinomas, and identical in both studies. However, there was lower concordance in the case of the acromegaly and of the functioning lactotroph PitNET in both series, with minor differences between them.Pre-surgical treatment for acromegaly with somatostatin analogues has been shown to reduce the levels of SSTR2 proteins and coulWhereas the effect of somatostatin on GH gene expression is unclear, dopamine suppresses PRL gene expression through the activation of intracellular signalling pathways in experimental animals. The maiThe non-functioning subtypes of the PitNET told a different story. In these tumours, the clinical endocrine syndrome did not corroborate the diagnosis of the patient. Thus, our gold standard was the IHC identification of the tumours. The concordance between molecular and IHC identification of the different subtypes of PitNET was very poor in the both series studied. Although we observed better concordance between molecular and IHC identification of the functioning subtypes than the previous series, the concordance did not improve in the non-functioning subtypes. With the exception of the silent corticotropinomas, moreover, the molecular-IHC concordance was different between the two series . One expSilent PitNET are pituitary tumours that immunohistochemically express specific pituitary hormones and cell specific transcription factors. So this type of tumours show ultrastructural features of a particular pituitary cell type . TherefoIdentifying silent PitNET variants seems to be important. SLT areOf particular importance, the molecular study will allow us to typify the PitNET of our series according to the new 2017 WHO classification of anterior pituitary tumours[However, as with the previous classification of anterior pituitary tumours, the new classification relies again on immunohistochemistry, and it will probably generate the same between-centre variability in the interpretation of the results. Although in this study we did not analyse the pituitary-specific transcription factors, detecting the dominant expression of the pituitary-specific hormone genes allowed us to identify the two subtypes of PH tumours with more confidence than with the IHC study. The Pit1-PH tumour corresponds to the previously termed ‘silent-subtype 3 adenoma’, which behaves with unusual aggression. These tThe molecular identification of PH PitNET entails a risk from contamination of the tumour with normal pituitary tissue, so it is very important that an expert pathologist refers to the material on which the molecular studies should be carried out.PitNET are one of the most common types of intracranial neoplasms. Their pet al. Like the previous study, a single researcher carried it out in a single centre. The series studied was specially selected on the basis of high-quality material provided by an expert pathologist and the availability of complete clinical, biochemical, radiological and IHC data for all patients.Strengths of our investigation include the reliability with which we reproduced the previous results published by Sanchez-Tejada On the other hand, the IHC took place in four participating centres, which makes an equitable comparison of both techniques difficult. The presence of gene expression does not guarantee the presence of protein expression. Therefore, despite the reliable information provided by the molecular study, its results should be complementary to the IHC study.This study reproduces, in an independent series, the results previously published by our group, enhancing the complementary role of the molecular study of the pituitary-specific hormone genes in the typification of PitNET subtypes. In addition, the two qRT-PCR techniques used are valid. Both techniques, TaqMan probes and SYBR Green primers, are suitable for the study of pituitary-specific hormone genes in PitNET. Although the molecular studies are unable to detect the densely and sparsely granulated variants of the PitNET, they help to reduce the number of null cell and plurihormonal tumours with an unequivocal clinical repercussion. Finally, we expect that the molecular study of the pituitary-specific transcription factors complements the IHC identification, ultimately improving the classification of PitNET subtypes.S1 Table(DOC)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.


Kabuki syndrome (KS) is a rare congenital anomaly syndrome affecting multiple organs. Two genes have been shown to be mutated in patients with KS: lysine (K)-specific demethylase 6A (KDM6A) and lysine (K)-specific methyltransferase 2D . Although the congenital clinical characteristic is helpful in diagnosis of the KS, there are no reports of specific findings in fetuses that might suggest the syndrome prenatally.In this study, we described a male patient with a novel KDM6A splicing in exon(exon4) and flanking intron(intron3)-exon boundaries characterized by congenital hydrocephalus which had never been reported before. The male patient had inherited the c.335-1G > T splice site mutation from his mother who had fewer dysmorphic features than the patient who displayed a more severe phenotype with multiple organ involvement. Our research suggests that congenital hydrocephalus may accompany KS type 2, which improve the knowledge on KS further more.Based on genetic and clinical features, suggest that the c.335-1G > T splicing mutation in KDM6A causing KS-2 disease. At least for this case, we suggest that congenital hydrocephalus is closely associated with KS type 2.The online version of this article (10.1186/s12881-018-0724-4) contains supplementary material, which is available to authorized users. Kabuki syndrome or Niikawa-Kuroki syndrome (KS) is a rare complex multi-system developmental disorder syndrome characterized by a stereotypical set of facial features, growth retardation, mild-to-moderate intellectual disability, organ malformation, hematological and endocrinological abnormalities –3, KS isThe use of the exome-sequencing strategy recently was able to identify KMT2D mutations as a major cause of KS, mutations in KMT2D were found in about 34–76% of KS patients , 10. In In this study, we investigated a three-month-old boy and analysed his clinical manifestations and symptoms, with microcephaly, micrognathia, distinctive craniofacial features, high arched palate, postnatal growth deficiency, intellectual disability, dysplasia of hip joint and congenital hydrocephalus. By consulting the relevant database, we found that hydrocephalus phenotype was not included in the previous clinical characterization in OMIM. Based on genetic feature, a novel splicing mutation c.335-1G > T) in KDM6A gene by Next-generation sequencing (NGS). Evidence of pathogenicity according to the ACMG, the mutation is splicing mutation, and no recorded in Normal public databases, and SPIDEX predict score is − 10.9497  results showed that enlarged lateral ventricles , weight (~ 2.9 kg) and head circumference (~ 33.5 cm) in accordance with the new WHO (2006) Child growth standards , suggest cm, weigThe results of cardiopulmonary and hepatolienal examination were normal. The patient had hypotonia, no pathological reflex. Liver and kidney functions, thyroid function, blood counts, electrolytes were normal. In addition, G-banding karyotyping showed that the patient had macroscopically normal chromosomes. Furthermore, heritage metabolic disease screening and antibody testing of maternal intrauterine infection were negative.Due to the clinical manifestations, imaging and laboratory tests results, it was suspected that the patient may be suffering from KS. Genomic DNA was isolated from peripheral-blood leukocytes, whole-genome CNV was obtained by NGS following the manufacturer’s protocol . Whole-genome analyses of CNV have not found definite pathogenic gene Fig. . All 19 KS is a rare monogenic disorder that is characterized by characteristic facial features, postnatal growth retardation, intellectual disability, immunological dysfunction, endocrinological and hematological abnormalities, and organ malformation –7. KS isMost of the published point mutations in KDM6A consist of nonsense mutations, small deletions, missense variants, and splice-site mutations. In addition, the published KDM6A mutations also included large deletions, large duplications/insertions and complex genomic rearrangement . KDM6A gIn conclusion, we identified a novel splice site mutation of KDM6A in a Chinese boy with KS2. We consider his congenital hydrocephalus manifestations to be the character of KS and may be informative and valuable for prenatal diagnosis. This is the first report of a KDM6A splice site mutation in a Chinese patient, and further research is needed to confirm the finding. Our study demonstrated that the congenital clinical characteristic was helpful in diagnosis of the KS.Additional file 1: Pathogenic evidence of the mutation in KDM6A gene. It shows the novel splicing mutation in KDM6A gene and the predication scores in various available predictive tools. We found PolyPhen-2, SIFT no predictiveed score, MutationTaster predictive was disease causing, GERP++_Predict is predicted Conserved result. SPIDEX predict score is − 10.9497, so the splicing mutation is likely pathogenic. (DOCX 15 kb)


Ganoderma lucidum is one of the most important fungi used for spirit production. Although this fungus affects the aromatic complexity of spirits, only a small number of studies have focused on investigating the influence of G. lucidum on the aromatic profile and colour of spirits. The aim of the research is to evaluate the influence of adding G. lucidum and herbal extract on final concentrations of volatile compounds and sensory quality of several distillates. In this study, distillates  were used to produce new spirit-based beverages with the fungus G. lucidum only, or with the fungus and herbal extract. Fifty-nine aroma compounds were identified by GC-MS. The aromatic profiles were strongly influenced by the primary aromas of the distillates, but the addition of G. lucidum and herbal extract enriched the volatile fraction of distillates with a range of ethyl esters, with a fruity and floral fragrance. Higher alcohols, 1-propanol, 2-isobutanol and isoamyl alcohol, were the most abundant volatile compounds in the analyzed distillates and spirits. The lightness of distillates was from 60.7 to 63.6, and with the addition of Ganoderma it significantly decreased to the range from 43.6 to 50.5. The addition of the fungus also increased the intensity of red and yellow colours. The Ganoderma spirits scored very highly in sensory evaluation (17.6–18.3), significantly better than the spirits without any additions (16.1–16.9).The multicomponent mixtures consisting of herbs and fungi are commonly used for the production of alcoholic beverages with potential health-promoting effects in many Asian countries. The medicinal fungus  Spirits are primarily a means of enjoyment and, as such, are commonly consumed due to their pleasant sensory characteristics and the relaxing effect of ethanol. The selection of raw materials used for spirit production encompasses a vast range of cultivated or wild plants worldwide, but strongly depends on local or regional tradition. The raw materials have an important influence on the physicochemical characteristics of spirit-based beverages  during 2010–2011 and identified 18 aroma compounds, the main being the alcohols 1-octene-3-ol, 3-octanol, 1-octanol, 2-ethyl-1-hexanol, which accounted for about 48% of the compounds responsible for the flavour. Furthermore, another study investigated the volatile compounds of the essential oil fraction produced by G. lucidum fruiting body -linalool 4.4, S-(+)-carvone 4.4, 2-pentylfuran 2.8, α-terpineol 2.7 and n-nonanal 2.3.In China and other countries of Asia, numerous fungi have been traditionally used to produce alcoholic beverages with potential health-promoting, disease preventing and medicinal qualities. However, multicomponent mixtures consisting of herbs and fungi are most commonly used for the production of such beverages  was employed to identify the specific compounds with the highest influence on the sensory quality depending on the type of used distillate.The aim of this study is to evaluate the influence of adding Ganoderma lucidum (W. Curt.: Fr.) P. Karst fruiting bodies were obtained from Jiangsu Alphay Bio-Tech Co. Ltd. . Fruiting bodies were separated from spores using brushes and then air-dried at 40 °C to constant mass. The fruiting bodies were prepared for extraction by cutting into pieces (about 1 cm).G. lucidum and fungus plus herbal extract. The following distillates were used: grain distillate produced by the company Ada Vrenje, Belgrade, Serbia; homemade plum distillate from the Aleksandrovac region of Serbia; grape distillate produced at the Radmilovac Experimental School Estate, Faculty of Agriculture, University of Belgrade, Serbia; and wine distillate, an industrial product from Vršački Vinogradi, Vršac, Serbia. All distillates were diluted with distilled water to reach an alcohol strength of 45% (V/V), chemically analyzed and then used to prepare the spirits with Ganoderma.Different distillates were used to produce new spirit-based beverages with only the fungus V/V) ethanol using a shaker  at 200 rpm and room temperature ((20±2) °C) in the dark. After extraction, the mixture was pressed with hydraulic press  to separate the liquid from the solids. The obtained liquid was filtered through filter paper  and stored in a dark place.The 44 plant materials  were purchased from the Institute for Medicinal Plant Research Dr Josif Pančić, Belgrade, Serbia (Ganoderma is presented in G. lucidum (40 g/L) was added to the distillates: grain distillate (GD), plum distillate (PD), wine distillate (WD), and grape distillate (GrD). Extraction was performed using a shaker  at 200 rpm and room temperature ((20±2) °C) in a dark place for 60 days. After the extraction, the products were filtered through a filter paper , and the obtained Ganoderma spirits were further analyzed. Then, 10 mL of herbal extract were added per L of each Ganoderma spirit. The types of herbs and their mass per volume ratios for herbal spirit production were chosen according to our previous experience  at 10 kPa and 45 °C to a volume of 2 mL. The samples were stored in a refrigerator (4 °C) until analysis.All samples of spirits were prepared using liquid-liquid extraction. An aliquot of 50 mL of each spirit was diluted with 100 mL of distilled water and then mixed with 20 mL of internal standard solution (methyl 10-undecanoate diluted in methylene chloride (0.01 mg/mL)) and 10 g of NaCl. Each resultant mixture was stirred on a magnetic stirrer  in closed flasks for one hour. After mixing, the organic layer was separated from the water layer. The upper organic layer was dried with Naet al. (m/z range from 40 to 350 atomic mass units (amu).Analysis of volatile compounds using GC-MS was performed according to method described by Tešević The identification was performed by comparing the mass spectrum of a compound with the Wiley 275 Mass Spectral Library  was added to the sample prior to liquid-liquid extraction. Flame ionization detector  was used for the integrations of all chromatogram peaks. The semi-quantitative concentrations of volatiles in the samples were calculated as follows:where S is a sample and IS is internal standard.The results of GC-MS analyses were expressed in milligrams of methyl 10-undecanoate equivalents per litre of analyzed spirits.Sensory quality rating was conducted in triplicate by a sensory panel that consisted of five expert judges, staff members of the Faculty of Agriculture, University of Belgrade, who were experienced in judging the quality of alcoholic beverages. All experts were male and their age profile ranged from 30 to 60. Three 2-hour training sessions were performed over a period of two weeks using the experimental spirits. Overall sensory quality was assessed by evaluating five selected sensory characteristics: colour, clarity, distinction, odour  and flavour , a* (red/green colour component) and b* (yellow/blue colour component).Colour measurements of brandies were obtained using a portable tristimulus Chroma Meter model CR–400  according to the procedure described by Pecić Sensory evaluation and colour measurements were conducted in triplicate. The analytical colour data and chemical data were subjected to one-way analysis of variance (ANOVA). Sensory data were analyzed by 3-way ANOVA that included ‘brandies’ as fixed factor, and ‘judges’ and ‘replications’ as random factors. Tukey’s HSD test was used to compare the mean values, with the level of statistical significance set at 0.05. Principal component analysis (PCA) on the correlation matrix was applied to raw GC, analytical colour, and sensory data, which had been standardized and averaged. The results for six identified groups of chemical compounds  were summed up to obtain GC data for individual volatiles, with a representative compound chosen from each group. All statistical analyses were performed using Statistica v. 12  and 2-methyl-1-propanol (isobutanol) were the most abundant , odour and flavour) of the distillates and spirits with G. lucidum improved some sensory characteristics of the analyzed brandies. The total sensory scores of all Ganoderma spirits were in the range from very good to excellent quality (17.6 to 18.3). The Ganoderma spirits based on wine or grain distillate received better scores than the Ganoderma spirit based on the homemade plum brandy. A distillate with a simple sensory profile is more suitable for the production of Ganoderma spirit than more complex bases, such as plum distillate.According to the obtained results, the addition of Ganoderma spirits as an attempt to enrich their sensory characteristics. The sensory qualities of the spirits with Ganoderma based on grape distillate and plum distillate were significantly improved after the addition of the herbal extract. The addition of the herbal extract had a positive effect on the flavour of all spirits with Ganoderma  to 8.1 . The a* values of the distillates  were negative, and it was noted by the panel that these spirits were characterized by light tones of green colour. The addition of fungus and herbal extract increased the intensity of the resultant spirits to amber shades, characteristic of old cognacs. The b* values were positive, and in the range from 2.8 (grain distillate) to 39.2 (grain spirit with Ganoderma). Hence, all analyzed spirits had different intensities of yellow colour. Based on these results, it can be concluded that the addition of G. lucidum and herbal extract increased the intensity of yellow, from golden to caramel. The L*, a* and b* values were significantly different for the wine distillate with Ganoderma, and the sensory experts described its colour as olive green with a shade of yellow as the background.The value of parameter PCA was applied in order to assess how the obtained spirits were grouped, taking into account the composition and content of aromatic compounds, colour attributes and sensory characteristics . PCA wasGanoderma and herbal extract), and spirits with lower concentrations of ketones . From each group of chemical compounds detected by GC-MS representatives  were chosen, and the influence of these volatiles cannot be explained within the obtained four-dimensional PC space. Higher contents of these volatiles were found in plum distillate, wine spirit with Ganoderma and plum spirit with Ganoderma and herbal extract on the upper side of the PC1–PC2 plot  on the sensory quality of the spirits with the fungus. The higher scores for odour and flavour quality were given to the spirits characterized by higher contents of esters and acetone such as grape and plum distillates with Ganoderma and herb extract, and lower contents of ketones such as grape, plum, grain and wine distillates with the fungus and herb extract. The addition of G. lucidum and herbal extract decreased the lightness of the spirits, and increased the intensity of yellow and red colours compared to the initial distillates. The obtained results have shown that the addition of G. lucidum and the herbal extract had a positive effect on the sensory quality of all spirits with Ganoderma. Ganoderma spirits could be potential innovative products for regional markets.According to the results obtained in the present work, the used distillates had a strong influence on the aromatic profile of the produced spirits. The chemical composition of the distillate bases had an important influence on the solubility of the components originating from


The choice of surgical technique in orthognathic surgery is based primarily on the surgical treatment objectives (STO), which is a fundamental component of the orthognathic treatment process. In the conventional orthodontics-first approach, presurgical planning can be performed twice, during the preorthodontic  and presurgical phases . Recently, a surgery-first orthognathic approach (SFA) without presurgical orthodontic treatment has been introduced and combined initial and final STO at the same time. In contrast to the conventional surgical-orthodontic treatment protocol that includes preoperative orthodontics for dental decompensations to maximize stable postoperative occlusion, the SFA potentially shortens the treatment period and minimizes esthetic concerns during the decompensation period because skeletal problems are corrected from the beginning. The indications for the SFA have been proposed in the literature, but no consensus exists. Moreover, because dental occlusion of the pre-orthodontic arches cannot be used as a guide for establishing the surgical treatment plan, there are fundamental limitations in accurate prediction of postsurgical results in the SFA. Recently, the concepts of postsurgical orthodontic treatment are continuously changing and evolving to overcome this inherent limitation of the SFA. The elimination of presurgical orthodontics can change the paradigm of orthognathic surgery but still requires cautious case selection and thorough discussion and collaboration between orthodontists and surgeons regarding the goals and postoperative management of the orthognathic procedure. The conventional orthognathic approach, or an orthodontics-first approach, involves orthodontic decompensation before surgery. This conventional approach aims to allow optimal surgical correction of skeletal deformities by defining the severity of any skeletal problems before surgery. However, skeletal deformities become more evident or even severely aggravated during the presurgical orthodontic phase, and this is a major complaint among patients, especially those with class III dentofacial deformities. The recently proposed “surgery first approach (SFA)” eliminates or minimizes such esthetic concerns during the presurgical orthodontic periods and also shortens the treatment period , 2. BecaHowever, previously published literature on the SFA had various methodologic shortcomings and did not utilize protocols based on scientific evidence or consensus from practitioners . TherefoThe definition of SFA is usually “orthognathic surgery without presurgical orthodontics” . HoweverThere are definitive advantages in the SFA compared to the conventional approach. However, there are currently five fundamental questions based on the previous studies , 9, 11 ais the final STO), which means that the presurgical orthodontics need to be accurately predicted, with no luxury to modify the STO based on the actual presurgical orthodontic changes as is the case in the conventional approach. Where there is arch width discrepancy, asymmetric transverse arch, or severe crossbite or deep bite, it is difficult to simulate the possible orthodontic movements that can address these problems. Therefore, accurate prediction of the occlusion after post-orthodontic treatment in the SFA is extremely challenging in such cases. Even though three-dimensional (3D) virtual occlusal setups have been utilized, it is difficult to accurately predict and simulate the occlusion after postoperative orthodontic treatment. Frequently, multi-segment osteotomies are planned in the SFA instead of simulating postsurgical orthodontic movements [In the conventional orthodontics-first approach, presurgical planning can be performed twice: during the preorthodontic  and presurgical phases . Therefore, the surgical simulation and planning can be modified at the final STO, based on the orthodontic changes made during that interval. However, for the SFA, the STO can be performed only once ” . Liou etAlthough the SFA does not involve presurgical orthodontics, fixed orthodontic appliances are frequently placed preoperatively to facilitate postoperative orthodontic treatment. To take advantage of locoregional or systemic acceleration of osteogenesis after surgery , it is iRegarding postoperative orthodontics, there can be several options for presurgical preparation for the SFA according to the literature , 39, 45:Since surgical hooks cannot be placed on light round or weak rectangular wires, additional maxillomandibular fixation (MMF) screws or anchor miniplates are frequently utilized. Passive adaptation of conventional rectangular stainless steel wires is not easy for patients with severe crowding or spacing Fig. . The autAnother concern in the SFA is bracket failure during surgery . BracketWhere there is occlusal interference before surgery, surgeons frequently adjust occlusion by eliminating the premature contacts or high points immediately before or during orthognathic surgery. While these occlusal interferences can be minimized by presurgical orthodontics, the SFA requires more frequent reduction of occlusal interferences during the surgery in comparison.Management of postoperative physical therapy or guidance of postoperative occlusion in the SFA requires greater attention because the occlusion is completely dependent on the surgical splint. When patients have limited, unfavorable occlusal contacts after surgery, proprioception from such occlusal contacts can induce unexpected mandibular positions from posturing. These might influence the long-term outcomes of the surgery. Wearing and adjusting the surgical splint postoperatively is an important step for a stable occlusion and long-term skeletal stability. When a significant occlusal discrepancy is anticipated after surgery, buildup of occlusal resin to stabilize the immediate postoperative occlusion should be strongly considered. Since the SFA cannot achieve optimal postoperative occlusion, the postoperative management in the surgery-first and conventional approaches are almost never the same. In the SFA, unfavorable orthodontic movements after surgery can accelerate the postoperative malocclusion more quickly . The useThe SFA quickly corrects skeletal deformities from the beginning and can shorten the total treatment time. Various indications for the SFA have been proposed, but there is no consensus. Moreover, because dental occlusion of the pre-orthodontic arches cannot be used as a guide for establishing the surgical treatment plan, there are fundamental limitations in accurate prediction of postsurgical results in the SFA. Recently, the concepts of postsurgical orthodontic treatment are continuously changing and evolving. The elimination of presurgical orthodontics can change the paradigm of orthognathic surgery, but the SFA requires careful case selection and thorough discussion with the orthodontist regarding the goals and postoperative management of the orthognathic surgical procedure. Therefore, the orthodontist’s decision on the orthodontic feasibility weighs heavily on the decision to plan using the SFA.


SLC6A3 gene encodes the dopamine transporter (DAT). The common 9R polymorphism produces higher DAT concentrations and consequently lower baseline dopamine than SLC6A3 wildtype. Whether SLC6A3 genotype modulates the effect of dopaminergic therapy on cognition in PD is not known. We investigated the effect of dopaminergic therapy and SLC6A3 genotype on encoding and recall of abstract images using the Aggie Figures Learning Test in PD patients. Encoding depends upon brain regions innervated by the VTA, whereas recall is mediated by widespread brain regions, a number innervated by the SNc. We found that dopaminergic therapy worsened encoding of abstract images in 9R carriers only. In contrast, dopaminergic therapy improved recall of abstract images in all PD patients, irrespective of SLC6A3 genotype. Our findings suggest that 9R-carrier PD patients are more predisposed to dopamine overdose and medication-induced impairment of cognitive functions mediated by VTA-innervated brain regions. Interestingly, PD patients without the 9R polymorphism did not show such an impairment. SLC6A3 genotype does not modulate the dopaminergic therapy-induced improvement of functions mediated by SNc-innervated regions in PD patients.In Parkinson's disease (PD), cognitive functions mediated by brain regions innervated by ventral tegmental area (VTA) worsen with dopamine replacement therapy, whereas processes relying on regions innervated by the substantia nigra pars compacta (SNc) improve. The  Parkinson's disease (PD) is a neurodegenerative disorder characterized by substantial dopamine-producing neuron loss in the substantia nigra pars compacta (SNc) with relative sparing of dopamine-producing neurons in the ventral tegmental area (VTA). The SNc principally supplies dopamine to the dorsal striatum (DS), comprising the bulk of the putamina and caudate nuclei. The ensuing depletion of dopamine to the DS produces the cardinal PD motor manifestations of rigidity, tremor, and bradykinesia . DopaminAlthough motor symptoms are uniformly improved by dopaminergic therapy in PD, distinct cognitive functions are dissimilarly affected by dopamine replacement therapy. Some cognitive functions are worsened whereas others are ameliorated or redressed by dopaminergic medication . FunctioOf importance to the current study, learning, in its various forms, is mediated by VTA-innervated regions such as the VS, hippocampus, and medial frontal cortex –9. This SLC6A3, is a membrane transporter protein that resorbs synaptic dopamine. SLC6A3 is abundant in the striatum, midbrain, and hippocampus, but scarce in the prefrontal cortex (O-methyltransferase (COMT). In the SLC6A3 gene, a 40-base pair variable nucleotide tandem repeat element exists, with 9-(9R) and 10-repeat (10R) forms being most prevalent , encoded by gene l cortex —where syrevalent . Recent revalent , 22 thatIn 9R carriers, increased expression of DAT, and consequently lower concentrations of dopamine at baseline, are expected to enhance the ratio between phasic, pulsatile, dopamine bursts related to events such as reward, positive feedback, or behavior, and tonic, basal dopamine release that occurs at rest. This enhanced signal-to-noise ratio is expected to result in more efficient signaling and potentially improved learning and performance. However, given that this superior performance is expected on the basis of lower tonic, basal dopamine, 9R carriers are predicted to be more susceptible to overdose effects of exogenous dopamine.In line with the hypothesis that 9R carriers have more efficient dopamine signaling and potentially improved performance, 9R carriers have been shown to have enhanced activity in bilateral striatum upon the reception of positive feedback , 24. DreSLC6A3 gene polymorphisms on cognition in PD patients has scarcely been investigated . Further, participants were excluded if they were abusing alcohol, prescription or street drugs, or taking medications such as donepezil, galantamine, rivastigmine, memantine, or methylphenidate. Participants were also excluded if they were known to have greater than mild-moderate depression (Beck Depression Inventory score above 30/63) or greater than mild-moderate anxiety (Beck Anxiety Inventory score above 30/63). They were also excluded if they had any other neurological illness. This study was carried out in accordance with the recommendations, and was approved by the ethics review board of both Health Sciences North , and the University of Western Ontario . All participants provided written, informed consent in accordance with the Declaration of Helsinki.Presence as well as severity of disease were assessed for all patients both on and off dopaminergic medication using the motor subscale of the Unified Parkinson's Disease Rating Scale (UPDRS) by a licensed movement disorders neurologist (PAM). All patients were treated with dopamine replacement medications such as dopamine precursors, L-3,4-dihydroxyphenylalanine (L-DOPA), and/or dopamine agonists. Table SLC6A3 gene was carried out according to a previously described method , and genomic DNA extracted using the MagNA Pure Compact instrument . Genotyping of a 40-base pair (bp) variable nucleotide tandem repeat  located in the 3'-untranslated region of the d method , 36 withGenotyping for the COMT c.472G>A  polymorphism was performed using a TaqMan allelic discrimination assay  using 50 ng of genomic DNA per reaction.SLC6A3 gene. As such, 44 PD patients were included in our subsequent analyses.One participant was excluded due to an inability to properly determine his/her genotype for the All participants performed two versions of the Aggie Figures Learning Test (AFLT) on two consecutive days , 37 Fig. PD patiIn each session, a set of 15 abstract symbols, List A, was presented to participants. These symbols were presented one at a time for 1,000 ms in the center of a computer monitor. Participants were instructed to try to remember as many of these symbols as possible. After the entire list had been presented, the participant was given 120 s to draw all the symbols that they could remember onto a piece of paper. This study-immediate recall procedure for List A was repeated five times in each version of the AFLT task , participants were asked to draw all the symbols that they could freely recall from Lists A and B Figure .The AFLT was scored by two researchers who were blinded to the identity of the participants  and session . A single point was awarded for each recalled item that could be unambiguously identified. Therefore, items were classified as correct if they had minor distortions in their shape or orientation. Any discrepancies in scoring between the two scorers were addressed such that an agreement was reached concerning scoring of these items.The difference in the number of correctly recalled items from the first and final study-immediate recall phases was used as our metric of memory encoding . That isWe used the total number of items recalled from List A after the 30-min delay divided by the total score achieved in the final study-immediate recall phase as our measure of memory retrieval, referred to as Weighted Recall. Unlike study-immediate recall phases, recall after delay is believed to preferentially index retrieval processes . FurtherSLC6A3 10R/10R vs. 9R-carriers) as the between-subject variable, and Session (ON vs. OFF) as the within-subject variable. Where warranted by significant interaction results, we followed up with subsequent one-way ANOVAs with Session (ON vs. OFF) as the within-subject factor to explore the simple effects of Session within Genotype.Encoding scores and Weighted Recall scores were used as dependent measures in separate 2 × 2 mixed-design analyses of variance (ANOVAs) with Genotype , suggesting that failure to find differences was not the result of lack of power.When examining the effect of SLC6A3 gene distribution did not deviate from Hardy-Weinberg equilibrium .Thirty participants were homozygous for the 10R allele and 14 participants were carriers of a single 9R allele and a 10R allele. No participants were homozygous for the 9R allele. The COMT rs4680 polymorphism on frontal dopamine .Additionally, due to the well-documented effects of the dopamine , we alsoSLC6A3 genotype (SLC6A3 10R/10R vs. 9R-carriers) as the between-subject variable, and Session (ON vs. OFF) as the within-subject variable  = 4.840, MSE = 13.182, p = 0.033], however. To better understand this interaction, we next examined Session effects for each of the SLC6A3 genotypes separately. For 9R carrier participants, there was a significant main effect of Session . For 10R/10R participants, there no main effect of Session (F < 1). In sum, we see worsening of memory encoding in the ON state  in the 9R carrier group only.We examined encoding scores in the AFLT in a 2 × 2 mixed ANOVA with SLC6A3 genotype (SLC6A3 10R/10R vs. 9R-carriers) as the between-subject factor and Session (ON vs. OFF) as the within-subject variable  = 4.515, MSE = 1.099, p = 0.040], reflecting better recall performance when on relative to off dopaminergic medication. The Session x SLC6A3 interaction was not significant (F < 1). In summary, we found that the administration of dopamine replacement medication improved recall scores in all PD patients. There were no differential effects related to SLC6A3 genotype.We examined Weighted Recall scores in the AFLT in a 2 × 2 mixed ANOVA with SLC6A3 genotype impacted cognition, response to dopaminergic therapy, or both in PD. In PD patients both on and off medication, we differentially assessed encoding of abstract images—a cognitive function mediated by VTA-innervated brain regions , therapeutic response (UPDRS ON-OFF), PD duration, or dopaminergic dosage as expressed by LED to explain differential effect of dopaminergic therapy on encoding.There were no main effects of SLC6A3 affects re-uptake of dopamine, particularly in striatum and hippocampus. Recent meta-analyses have concluded that expression of SLC6A3 9R allele is higher than the 10R-homozygotes  cognitive functions mediated by VTA-innervated brain regions vs. DS, and (b) response to dopaminergic therapy in PD. Replicating our previous findings (improved recall performance in PD patients irrespective of SLC6A3 genotype, whereas it impaired encoding, but only for 9R-carriers. This pattern of findings is in keeping with the view that encoding is mediated by dopamine-replete VTA-innervated brain regions, such as hippocampus and VS, whereas recall is mediated by broad brain regions including the significantly dopamine-deplete DS and its cortical partners. These results indicate that whereas dopaminergic therapy benefits DS function in all PD patients irrespective of SLC6A3 genotype, 9R-carrier status predisposes to dopamine overdose of VTA-innervated brain regions. We speculate that higher SLC6A3 expression in 9R allele carriers, and consequently lower basal dopamine, yields a more optimized signal-to-noise dopamine ratio that is more vulnerable to disruption by exogenous dopamine. This is in comparison to 10R homozygotes who are adapted to higher and more variable baseline, tonic dopamine. These results suggest that 9R-carrier status predisposes to dopamine overdose.To our knowledge, this is the first investigation of the effect of findings , dopaminPM, RK, KS, BR, and NH designed the experiment. BR, AA, AM, NH, US, RK, and RT acquired and analyzed data. BR and PM wrote the manuscript. All authors edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Kaposi’s sarcoma (KS) is one of the most common HIV-associated malignancies in sub-Saharan Africa. Worldwide, the availability of antiretroviral therapy (ART) has improved KS survival. In resource-rich settings, survival has also benefited from chemotherapy, which is widely available. Little is known, however, about the epidemiology of chemotherapy use for HIV-associated KS in resource-limited regions such as sub-Saharan Africa.We identified all patients newly diagnosed with HIV-related KS from 2009 to 2012 in the 26-clinic AMPATH network, a large community-based care network in Kenya. We ascertained disease severity at diagnosis, frequency of initiation of chemotherapy, and distribution of chemotherapeutic regimens used. Indications for chemotherapy included AIDS Clinical Trial Group T1 stage and/or “severe” disease defined by WHO KS treatment guidelines.> 3 chemotherapy indications at time of diagnosis had a 2.30 (95% CI 1.46–3.60) increased risk of rapid chemotherapy initiation (within 30 days of diagnosis) compared to those with only one chemotherapy indication (p < 0.001). Initial regimens were bleomycin-vincristine (78%), adriamycin-bleomycin-vincristine (11%), etoposide (7%), and gemcitabine (4%).Of 674 patients diagnosed with KS, charts were available for 588; 61% were men, median age was 35 years, and median CD4 at KS diagnosis was 185 cells/μl. At time of diagnosis, 58% had at least one chemotherapy indication, and 22% had more than one indication. For patients with a chemotherapy indication, cumulative incidence of chemotherapy initiation (with death as a competing event) was 37% by 1 month and 56% by 1 year. Median time from diagnosis to chemotherapy initiation was 25 days (IQR 1–50 days). In multivariable regression, patients with A substantial fraction of patients with KS in East Africa are diagnosed at advanced disease stage. For patients with chemotherapy indications, nearly half did not receive chemotherapy by one year. Liposomal anthracyclines, often used in resource-rich settings, were not first line. These findings emphasize challenges in East Africa cancer care, and highlight the need for further advocacy for improved access to higher quality chemotherapy in this setting. In many areas of Africa affected by the HIV epidemic, HIV-associated malignancies are among the most common cancers in the overall population . For exaMuch of the explanation given for the high mortality of HIV-related KS in Africa has been attributed to advanced stage at presentation, but the role of therapy must be considered as well. There are two established therapies: ART and chemotherapy. ART is indicated for all HIV-infected patients with KS regardless of cancer severity , and canTo address our limitations in knowledge about the epidemiology of chemotherapy use for HIV-related KS in Africa, we investigated all newly diagnosed cases of KS in a community-based HIV care program in East Africa. We determined the prevalence of indications for chemotherapy at the time of diagnosis, frequency of initiation of chemotherapy, drug regimens used, and factors associated with chemotherapy initiation.Measurements below). In a cohort analysis, we then observed subsequent clinical course, including any therapy received, for 12 months following KS diagnosis.Via medical record review, we identified all patients newly diagnosed with HIV-related KS from 2009 to 2012 in the Academic Model Providing Access to Healthcare (AMPATH) consortium in Kenya. We determined indications for chemotherapy at the time of KS diagnosis .To identify all KS cases over the specified time period, we reviewed the clinic network’s electronic database as well as pathology records, and then followed up with detailed paper chart review. We included all patients living with HIV We ascertained KS disease severity at time of diagnosis and subsequent KS-specific treatment and survival through chart review. HIV/primary care charts, oncology charts, chemotherapy infusion records, and inpatient charts were retrieved for each participant. Paper charts were sought on three separate occasions before being deemed unobtainable. Record abstraction was performed by trained clinical officers using a structured chart review form. Double data entry was performed, and differences were reconciled by the principal investigator (PI).KS severity at time of diagnosis was based on any symptom recorded within the first 30 days of KS diagnosis. Information on weight loss, diarrhea, night sweats, fever, Eastern Cooperative Oncology Group (ECOG) performance status, extent of cutaneous KS, lymphadenopathy, oral disease, GI KS, pulmonary KS, ulceration, and edema was extracted from the charts. We considered chemotherapy indications to be the presence of at least one AIDS Clinical Trials (ACTG) T1 qualifying criteria   or the pTime zero was set as the earliest date the clinician had information about the KS diagnosis and therefore could potentially act on it. When a biopsy was performed, the date of the receipt of those results to the clinician  was used for time zero. If a patient had both a clinical diagnosis date and a biopsy diagnosis date, the earlier of the two dates was used to establish time zero. Observation was continued from time zero to the initiation of chemotherapy or death for 12 months. Patients not known to be dead or to have started chemotherapy were administratively censored at last known date alive.Cumulative incidence of chemotherapy initiation with death as a competing event was calculated using the Aalen-Johansen estimator . PatientOf 674 patients diagnosed with KS during the study period, 588 (87%) had charts available for extraction and were included in the study. The majority of diagnoses were made on clinical grounds alone, while 266 (45%) had biopsy-confirmed KS. Most 61%) were men and median age was 35  were on ART at time of KS diagnosis, with 112 (19%) on ART for > 60 days at time of diagnosis. The ART regimen administered prior to 2011 in Kenya included stavudine (d4T) or zidovudine (AZT), plus lamivudine (3TC) and efavirenz (EFV) or nevirapine (NVP) [Median proximal CD4 count was 184 cells/mmne (NVP) . In 2011ne (NVP) .p = 0.004).The most common symptoms at time of KS diagnosis were non-localized skin disease (e.g. skin disease that involved more than one anatomic location), edema, and oral lesions. Relatively few had suspected or confirmed gastrointestinal or pulmonary KS  was 27% by 1 month, 43% by 3 months, 48% by 6 months, and 50% by 1 year . Of the remaining patients, 11% received doxorubicin in addition to BV, while 7% received etoposide and 4% received gemcitabine. No patients received paclitaxel or liposomal anthracyclines as first line chemotherapy. The most common adverse effects included peripheral neuropathy (43.8%), neutropenia (25%), and gastrointestinal (GI) upset (25%).The most common first-line chemotherapy regimen in this Kenyan cohort was bleomycin-vincristine (BV), at 78% . Similarly, in a sensitivity analysis restricted to patients with biopsy proven KS only, patients with three or more chemotherapy indications had a 2.12  times risk of rapid chemotherapy initiation, though this did not quite reach statistical significance due to the smaller sample size of patients that had biopsy-proven disease (p = 0.07).We present two adjusted models for determinants of chemotherapy initiation within 30 days of KS diagnosis Table . Model 1< 50 was not associated with rapid chemotherapy initiation compared to those with the highest CD4 > 350. However, moderately low CD4+ T cell count of 51–200 was associated with a modest increase in chemotherapy initiation . Being on ART at time of diagnosis was not associated with chemotherapy initiation within 30 days of KS diagnosis.In the multivariate models, neither age nor gender were significant predictors of chemotherapy initiation. Restricting to biopsy proven KS cases did not change this result. None of the evaluated socioeconomic indicators (Model 2) were associated with chemotherapy initiation. Notably, there was limited variability in the socioeconomic status of patients using these Ministry of Health supported clinics; 72% either received no education or primary school only, and 87% did not have running water in their homes. Having a very low CD4+ T cell count Among patients with an indication for chemotherapy at time of KS diagnosis, we evaluated the joint occurrence of chemotherapy and death over time Fig. . By 12 mKS mortality in sub-Saharan Africa is high, which led us to investigate the use of chemotherapy for treatment of KS in this setting. Among patients in a primary care HIV cohort in Kenya, just over half of patients with KS who had an indication for chemotherapy received it by the end of 1 year. Patients with the most severe disease were the most likely to receive prompt treatment within 30 days of KS diagnosis . However, a substantial group of patients (44%), who seemingly had an indication for chemotherapy based on the severity of their KS disease, did not receive it within 1 year. Given the high mortality of KS in sub-Saharan Africa [Prior studies on KS treatment with chemotherapy in sub-Saharan Africa have focused on outcomes of particular chemotherapy regimens, , 25–27 bSimilar to other resource poor settings, most patients in this Kenyan setting were diagnosed with KS when their disease was already at advanced stage, and not all patients had histologically confirmed disease , 31, 32.The most common first line chemotherapy regimen in this setting was bleomycin plus vincristine. Liposomal anthracyclines  and paclitaxel, which are first-line medications in resource-rich settings, were not used as initial therapy in any patient. Liposomal anthracyclines are recommended over BV when available in both the WHO and the National Comprehensive Cancer Network (NCCN) guidelines , 13, altPatients with more chemotherapy indications (e.g. more advanced disease stage at time of diagnosis) are more likely to achieve rapid chemotherapy initiation within 30 days of diagnosis. That the most ill patients are being prioritized for chemotherapy is reassuring. The lack of impact of socioeconomic factors on chemotherapy initiation was initially surprising, since we hypothesized that patients who reside farther from clinic with less education and fewer resources would have a harder time accessing care. There are several possible reasons for this observation. This lack of effect may be due to the relative homogeneity of this population of care seekers, who are overwhelmingly economically disadvantaged and have at most a primary school education, where small variations in socioeconomic status may not impact access to chemotherapy. It is also possible that we need more sensitive socio-economic measures to delineate this effect. Alternatively, the community outreach programs of AMPATH, where chemotherapy is supplied to two other community clinics outside of the main hospital, may have helped to mitigate the effect of socioeconomic status on access to chemotherapy.This study had several limitations. First, we identified people only after they had been diagnosed with KS. Therefore, we did not account for patients in the community who either did not seek care, or sought care but did not receive a formal KS diagnosis. Therefore, it may overestimate the true proportion of KS patients that received chemotherapy. Second, we were unable to locate paper charts for 13% of patients over the study period who were diagnosed with KS. These patients were excluded from the study since we were unable to ascertain whether or not they had received chemotherapy. Thirdly, severity of KS at time of diagnosis was limited to what clinicians recorded in the patients’ charts during routine clinical care. Record keeping was often incomplete. Absence of a symptom (e.g. a pertinent negative) was rarely recorded. Therefore, when a particular symptom was not documented, we made the assumption that the patient did not have that symptom. It is possible, therefore, that we underestimated the severity of disease at diagnosis. In particular, WHO severe disease was likely to be underestimated, since it was not common practice to record whether the disease was affecting the patient’s functional status.Due to the retrospective nature of this chart review and incomplete paper records, we were not able to ascertain reasons that eligible patients did not start on chemotherapy. Literature suggests that for other cancers in sub-Saharan Africa affordability , 43, traRegarding generalizability, this Kenyan cohort consists of the general population receiving HIV primary care in a resource-poor sub-Saharan Africa setting. However, the AMPATH program does have access to enhanced oncology services, including a public cancer center with routine outreach clinics and an IeDEA-sponsored KS biopsy service. Therefore, both diagnosis and treatment in the setting may be enhanced compared to other public sector settings elsewhere in sub-Saharan Africa .In conclusion, we found that 44% of patients with KS in this Kenyan setting who may have needed chemotherapy at the time of diagnosis did not initiate this treatment. The subset of patients who successfully initiated chemotherapy did, for the most part, receive it in a timely manner. Due to the retrospective nature of this study, we could not further elucidate the reasons for treatment non-initiation, or distinguish between patient and provider factors. Prospective evaluation is needed to assess barriers to chemotherapy from both patient and provider perspectives, so that appropriate interventions can target treatment initiation. The chemotherapy regimens used at the time of the study, and what is still routinely available in Kenya, are not equivalent to liposomal anthracyclines, which are first line in resource-rich settings. International advocacy for more efficacious, affordable, and accessible chemotherapy in sub-Saharan Africa is critical, and improved outcomes could encourage other patients to seek out and initiate chemotherapy.


Antimicrobial resistance (AMR) is an increasing threat to global public health that is largely exacerbated by the overuse and misuse of antimicrobial medicines. As the largest antimicrobials producer and user in the world, China has a critical role to play in combatting AMR. By examining Chinese news articles and policy statements, we aim to provide an authentic understanding of public discourse in China on AMR.A search was conducted using two of the most comprehensive digital libraries for Chinese news media documents. Chinese policy documents were retrieved from official Chinese government websites. Records from June 2016 to May 2017 were included. Grounded theory was used to analyze included records, and we followed an iterative thematic synthesis process to categorize the key themes of each document.Across 64 news articles, most articles delivered general knowledge about AMR and debunked AMR-related myths, explored the implications of AMR-relevant policies, and discussed the misuse of antimicrobials in the agricultural sector. All policy documents provided guidance for healthcare workers, encouraging them to better manage antimicrobial prescriptions and usage.While the Chinese media actively educates the public on strategies for AMR prevention, certain news articles risk misleading readers by downplaying the hazards of domestic AMR issues. Further, although several national policies are geared towards combatting AMR, the government faces difficult challenges in overcoming public misconceptions regarding antimicrobial use. Records from the regional level should also be examined to further explore China’s public discourse on AMR.The online version of this article (10.1186/s41256-019-0097-z) contains supplementary material, which is available to authorized users. Antimicrobial resistance (AMR) occurs when microorganisms evolve to withstand antimicrobial medicines. This phenomenon hinders the effectiveness of antimicrobials in the prevention and treatment of a multitude of infectious diseases. Although AMR is a process that may naturally occur, the overuse and misuse of antimicrobial drugs accelerates this development . AMR is Results from a survey conducted by the Chinese Academy of Sciences show that total antibiotic usage in China in 2013 was approximately 162,000 tons, which consists of 52% of usage in animals and 48% in humans, making China the largest antibiotics producer and user in the world [Staphylococcus aureus (MRSA), which is a bacterium that may cause life-threatening infections due to its resistance, has a mean prevalence of 80.4% in Shanghai and 50.4% in China as a whole [The widespread use of antimicrobials in China has surely exacerbated the country’s AMR problem. Bacteria that confer resistance to colistin, a last-resort antibiotic for bacterial infections, has been identified in animals and samples from infected Chinese inpatients as of 2015 [ a whole , 7. ThesIn recent years, China has joined global AMR efforts by implementing a series of national strategies to address AMR, including its National Action Plan (NAP) to Contain Antimicrobial Resistance (2016–2020) released in August 2016 . The NAPDespite China’s increasing engagement with AMR, we know of no published articles that have analyzed public discourse on AMR as reflected by news media articles and policy statements in China. These original documents in Mandarin Chinese are inaccessible to most researchers who are not proficient in the language, and the lack of assessment of these records hinder many from gaining a more authentic understanding of China’s current public discourse on AMR. Combined with China’s massive domestic population and its role as a global power, there is a clear need to understand how this issue is discussed and framed in both Chinese popular discourse, as reflected by news media articles, and political discourse, as reflected in policy statements.This study qualitatively surveys the ways in which AMR is discussed in two types of Chinese documents that are often not translated to English: news media articles and policy statements. We then discuss challenges that China faces in acting on AMR and recommend strategies for how China can strengthen its efforts against AMR.We conducted a systematic search of news media documents in the China Core Newspapers Full-Text Database and Global Think Tanks, which are part of the China National Knowledge Infrastructure (CNKI) and Wanfang, respectively. Both the CNKI and Wanfang are digital libraries that consist of sub-databases for different types of publications, including newspapers, magazines and gazettes. For policy statements, we searched official Chinese government websites, which provide content directly from the State Council of the People’s Republic of China, the National Health Commission of the People’s Republic of China, and other relevant national-level ministries. These sources were recommended by two librarians at Nankai University in China.All sources were searched using the following phrases in Chinese: “抗生素滥用”或者“抗菌素滥用”, “抗生素耐药性”或者“抗菌素耐药性”, and “超级细菌, 耐药细菌”. These phrases were developed and refined by conducting pilot searches and consulting with librarians at Nankai University. We searched each phrase individually when using systems that do not offer advanced search functions, such as government websites.All records included in our analysis were published and analyzed in Mandarin Chinese. This allowed us to avoid the biases or misinterpretations that may occur in analyzing translated works. We included only records from 1 June 2016 to 31 May 2017. This one-year period was selected as it captures a period of substantial international attention on AMR and discourse on AMR. This time period follows the release of the final report by the UK Review on Antimicrobial Resistance led by Jim O’Neill , and capGiven the top-down nature of domestic policy making in China, we excluded records published by provincial- or local-level newspapers and governments and focused on national-level news articles and policies. We further excluded any news articles that reported only basic science discoveries; we did include records that discussed AMR in a societal context, such as those that emphasize the political or economic implications of AMR.We followed a grounded theory approach to analyze included documents, which involves an inductive process of extracting and categorizing data from the records to generate new themes . Prior tWe conducted a three stage thematic synthesis: the first stage involved coding each document based on its key messages ; the secn = 100 and n = 61, respectively). Among the 114 news articles, 64 met our inclusion criteria and were included in our analysis, as were the five identified policy statements. Our thematic synthesis identified 14 different conceptual themes, which are described in Table We identified 114 news articles published during our study period, which was more than in the previous and following years  were aimed at China’s domestic population. The five policy documents, and eight news articles, all shared the theme of professional awareness, emphasizing that physicians and medical institutions need to better manage antimicrobial prescriptions and usage. One policy established an Expert Committee on Clinical Application and Evaluation of Antibiotic Resistance, which is tasked with providing relevant research and policy recommendations. Only the NAP – China’s most comprehensive policy targeting AMR – touches upon efforts for global collaboration and management of AMR in agriculture. The NAP also recommends promoting rational use of antimicrobials through media awareness campaigns, which closely aligns with the media’s emphasis on public awareness and education.The future directions presented in several news articles are also consistent with the goals and strategies stated by the NAP. The media articles provided a general introduction to AMR and the strategies recommended in the NAP to combat AMR in both healthcare and agricultural sectors, with two articles published following the release of the NAP incorporating five different AMR themes.Many of the 29 news articles focusing on public awareness and education were aimed at counteracting the public’s fear of AMR, and quoted scientific experts to increase their credibility. However, many of these experts were unnamed and it was therefore difficult to assess the validity of their claims. Importantly, there were ten news articles that attempted to combat panic among the Chinese public that resulted when researchers found numerous antibiotic-resistant genes in Beijing’s smog . Many ciWe found that news articles with a policy theme discussed the challenges of implementing AMR-related policies and criticized attempts to provide a quick solution to the complex issue. One example relates to the intravenous (IV)-ban policy discussed by 11 of the 16 news articles that had a policy focus. Although it has yet to be formally established at the national level, the policy of banning all IV drug infusions at higher level outpatient clinics has been implemented in several provinces. The news articles that discussed this policy often concluded that simply banning the use of IV antibiotics is not enough to overcome AMR, as fundamental problems such as the lack of standardized physician training on antimicrobial prescribing are not being addressed. However, these articles acknowledged that stricter regulation of IV prescriptions was needed and most encouraged similar policies to be implemented across the country. China has reportedly consumed 10.4 billion IV infusion bottles in 2009, which averages to eight bottles used per citizen and is much higher than the global average . Other pTwelve articles focused on the impact of agricultural use of antimicrobials on AMR and addressed concerns over antimicrobial misuse in livestock breeding. These articles also urged the government to take more specific actions to combat AMR in the agricultural sector. Two articles target the public’s discontent over antibiotic-fed chickens. Many Chinese citizens accused American fast-food chains of employing a double-standard; these companies have committed to using antibiotic-free chickens at their US stores but have yet to do so at their international stores, including those in China. The Chinese media articles we identified attempted to reassure the public that antibiotics were beneficial to public health when used responsibly during livestock breeding and that “antibiotic-free” merely means that they do not use antibiotics important in human medicine. These articles also shared the theme of public awareness and education. Additionally, many news articles claimed that the severity of antimicrobial overuse in agriculture was comparable to that in clinical settings, but only described two specific actions that the government had taken in this sector: the establishment of a new working standard to detect antibiotic levels in animal manure, and a national research project on livestock pathogen resistance and testing.Our results indicate that Chinese news media articles generally aligned their focus with that of government policies, suggesting that the risks of AMR are portrayed in a way that the national government approves. Given tight media controls, we found evidence that the Chinese government likely uses its country’s news media to help manage public fears associated with health threats like AMR. In some cases, the news articles analyzed in this study appear to have employed strategies such as distorting or omitting scientific data, which may hinder Chinese citizens to readily access information that can help them confront daily issues or make informed decisions, such as whether to eat antibiotic-fed livestock. Nevertheless, Chinese news media also provides useful advice regarding AMR prevention that citizens should implement in their daily lives, such as avoiding inappropriate reliance on antimicrobials for prevention and treatment of infections when not clinically indicated. AMR prevention methods suggested by these news articles match recommendations by the WHO and reiterate the global severity of AMR . In addiThe Chinese news media appears to be frequently used as a tool for managing public fear in the face of health threats. The Chinese media accurately depicts the consequences of AMR when it is portrayed as a global problem, but takes on a different tone when discussing contentious, internal AMR issues in China. This is unsurprising given strict media controls in China, with Freedom House ranking China last out of 65 countries for press freedom in 2015–2016 . SpecifiThe New York Times, have criticized Chinese news articles that debunk the smog myth for downplaying the risks of antibiotic-resistant genes, or suggested that health advice provided by state news outlets was merely used to “assuage the worries of locals” [The New York Times quotes skeptical internet users who urged the Chinese government to focus on developing strategies to combat AMR rather than reassuring the public. Internationally, there are concerns about the validity of claims made in Chinese news media articles. For instance, Joakim Larsson and his team of researchers sampled various environments worldwide for antibiotic resistance genes, and found that these genes were present in air samples from a Beijing smog event [An interesting example is the Chinese news media’s treatment of the smog myth, or the public’s fear that antibiotic-resistant genes in Beijing’s smog will cause significant harm in humans. Several international publications, including  locals” –26. The og event . Chineseog event . Nonetheog event , 27. Thiog event .Another example of the Chinese media’s attempt to manage public fear is its response to the public’s disapproval of antibiotic-fed chickens, which are being used in Chinese restaurants. These same restaurant chains have eschewed antibiotic-fed chicken in US restaurants. Chinese news media articles may be accused of ignoring the complexity of agricultural AMR, as they emphasize that preventative antimicrobials, which can promote animal growth, are in fact beneficial to maintaining the animals’ health when used in moderation. This issue has been controversial globally, with some researchers, and members of the agricultural industry, arguing that agriculture does not significantly contribute to the development of AMR in humans . MeanwhiThe news media’s treatment of AMR was different in articles on regulating the use of IV antibiotics. Here, we found that by interviewing frontline healthcare providers, the news media delivered an alternative perspective on policy implementation, and emphasized the challenges in regulating antibiotics, thereby affording readers a better understanding of the practical implications of different government policies.Hospitals in China are widely known to overuse IVs, even though IV is shown to have a greater risk and severity of error than other drug delivery methods , 31. SevFaced with an enormous population and a complex healthcare system, the challenges identified above are difficult for the Chinese government to overcome. Nonetheless, the Chinese news media can continue to heighten public and professional awareness of AMR, as well as educate citizens about strategies that can prevent AMR’s spread. Current research efforts to identify evidence-based strategies for government interventions on AMR may support China’s efforts to protect public safety and effectively achieve the goals of its NAP . The ChiBased on the significant number of news articles examining the impacts of antimicrobial use in agriculture, the Chinese government could take more concrete steps towards managing antimicrobial usage in agriculture. Further, with its significant influence, China could assume a global leadership role in tackling this challenge – by role modeling progress domestically and supporting collective action globally.Finally, despite the increase in discussions of AMR at the international level, we found that only four Chinese media articles and one policy document emphasized the need for global collaboration. As a global collective action problem, stemming the tide of AMR will require concerted action and coordination by all countries . The samThis study has four key strengths. First, we conducted a systematic search of different databases and websites, capturing 365 days of activity at a time when there was significant focus on AMR globally. Second, we compared two distinct types of documents, which allowed us to analyze and compare both the government’s stated policy and the way AMR is portrayed in popular discourse. Third, we followed a rigorous qualitative methodology that involved three stages of iterative coding, and triangulation between two types of data–news media articles and policy statements. Fourth, and most importantly, we directly assessed records in Chinese rather than relying on translations, which allowed us to include many Chinese news articles and lesser-known policy statements that do not have translated versions available. These are valuable sources of information that provide a more comprehensive depiction of popular and political discourse on AMR in China.There are three main limitations of this study. First, we analyzed articles for a one-year period; it is likely that public discourse has shifted over time, but we are unable to track long-term change in the current study. Second, we focused on public discourse at the national level only, excluding discourses in individual municipalities and provinces in China – some of which are more populous than other major countries. Different provinces in China may have unique challenges and perspectives regarding antimicrobial use that are not addressed in national-level news articles and policy statements. Third, while we searched two large databases of news-media articles and official Chinese government websites, we recognize that some media and policy sources may not be indexed in these locations, and that much of popular discourse in China occurs via web-media, such as WeChat, that may systematically differ from discourse in newspapers ; howeverNews media articles have played a constructive role in attempting to educate the public about AMR, although concerns have been raised about news articles that provide false reassurance of public health and safety. National policies reflect the Chinese government’s engagement with global action on AMR. However, efforts to implement these policies must overcome various challenges such as public misconceptions regarding antimicrobial use. Future initiatives could target agricultural AMR and global collaboration of stewardship.To further explore Chinese discourse on antibiotics, changes or updates in popular and political discussions about AMR should be examined and compared to what is seen in other countries. Municipal and regional level documents should also be analyzed to account for the fact that China is a diverse country with many perspectives, challenges and opportunities for addressing AMR.Additional file 1:Thematic Synthesis of News Media Articles and Policy Statements.xls. This MS-Excel spreadsheet provides the detailed qualitative analysis of each news media article and policy statement conducted in this study, as described in the Methods section. It also lists the source (database), publisher, publication date, access date, title, and URLs of all analyzed records. (XLSX 142 kb)


Efforts to investigate whether cDC1 might orchestrate immune defenses against cancer are ongoing, thanks to the recent blossoming of tools allowing their manipulation in vivo. Here we are reporting on these studies. We discuss the mouse models used to genetically deplete or manipulate cDC1, and their main caveats. We present current knowledge on the role of cDC1 in the spontaneous immune rejection of tumors engrafted in syngeneic mouse recipients, as a surrogate model to cancer immunosurveillance, and how this process is promoted by type I interferon (IFN-I) effects on cDC1. We also discuss cDC1 implication in promoting the protective effects of immunotherapies in mouse preclinical models, especially for adoptive cell transfer (ACT) and immune checkpoint blockers (ICB). We elaborate on how to improve this process by in vivo reprogramming of certain cDC1 functions with off-the-shelf compounds. We also summarize and discuss basic research and clinical data supporting the hypothesis that the protective antitumor functions of cDC1 inferred from mouse preclinical models are conserved in humans. This analysis supports potential applicability to cancer patients of the cDC1-targeting adjuvant immunotherapies showing promising results in mouse models. Nonetheless, further investigations on cDC1 and their implications in anti-cancer mechanisms are needed to determine whether they are the missing key that will ultimately help switching cold tumors into therapeutically responsive hot tumors, and how precisely they mediate their protective effects.Dendritic cells (DCs) are endowed with a unique potency to prime T cells, as well as to orchestrate their expansion, functional polarization and effector activity in non-lymphoid tissues or in their draining lymph nodes. The concept of harnessing DC immunogenicity to induce protective responses in cancer patients was put forward about 25 years ago and has led to a multitude of DC-based vaccine trials. However, until very recently, objective clinical responses were below expectations. Conventional type 1 DCs (cDC1) excel in the activation of cytotoxic lymphocytes including CD8 Indeed, tumors escape host immune defenses not only through decreasing their intrinsic immunogenicity but also by shaping a specific immunosuppressive microenvironment , 2. Exogignature . “Cold” ignature . They ar+ cDCs as well as the CD103+CD11b− cDCs that reside in the parenchyma of non-lymphoid tissues and, once matured upon activation, can migrate to the draining lymph nodes. In humans, cDC1 correspond to the CD141 (BDCA3)high CD11b−/low cDCs. Both mouse and human cDC1 express specifically the chemokine receptor XCR1 and selectively the C-type lectin endocytic receptor CLEC9A high CD11b+/high cDCs. For a very long time, MoDCs were the only DC type that could be produced in vitro, in high numbers and under clinical-grade conditions  are the most potent Ag-presenting cells, with a unique efficacy for priming naïve T cells and inducing their functional polarization. They are more generally in charge of orchestrating the expansion and functions of T and natural killer (NK) cells in lymphoid and non-lymphoid tissues. Many clinical trials have been performed over the last 25 years to attempt harnessing DC functions for boosting protective antitumor CTL responses in cancer patients –8. A relr CLEC9A , 13. Mounditions , 8. They in vivo . For exa in vivo . Thus, min vivo, it should be specifically and efficiently manipulated in mice. Different mutant mouse models have been generated to either deplete DCs, or inactivate candidate genes in DCs, as recently reviewed (Itgax-Cre targeting CD11c+ cells and Xcr1-Cre targeting cDC1). Constitutive depletion models have been generated using two types of strategies. The first corresponds to the knock-out of a transcription factor shown to be crucial selectively for the development/homeostasis of cDCs (Zbtb46) or cDC1 (Batf3) (DTA) selectively in DCs  selectively in DCs .To determine whether and how a given type of immune cells plays a non-redundant role in antitumor immunity reviewed , 19. Herreviewed   . The secItgax, is expressed by other immune cell types, including some that play critical roles in anti-tumor immunity, such as NK cells, effector memory CTLs, intraepithelial lymphocytes (IELs), plasmablasts, and subsets of monocytes or macrophages  in knock-out mice  gene, but these models also target mast cells  according to their immunogenicity, their cDC1 infiltration, maturation and phenotype, and the characteristics of the antitumor CTL response.“Hot” tumors are characterized as strongly infiltrated by effector CTLs. They are spontaneously controlled by the immune system . They in“Warm” tumors express tumor neoAg and are infiltrated by cDC1 and CTLs  Right. E“Cold” tumors are weakly immunogenic and poorly infiltrated but induce some level of adaptive immunity  Top. In per se, are not infiltrated by T cells and fail to induce immune responses , i.e., CTL ACT. Second, we will discuss mAb immunomodulation (mAIM) to block checkpoint receptors on CTLs or NK cells , a good prognosis of a higher tumor infiltration by cDC1 has been documented independently by 4 studies , 66, 182Similar analyses were performed for other types of cancer. For head and neck squamous cell carcinoma (HNSC) and lung adenocarcinoma (LUAD), a higher expression of the cDC1 transcriptomic signature in tumor was also associated to a better clinical outcome by at least two independent studies , 62, 80.All of the above studies suggest that, in a variety of human cancers, intra-tumoral cDC1 abundance correlates with a better clinical outcome. However, further studies are required to confirm these results, to extend these types of analyses to other types of cancer, and to deepen our understanding of the underlying mechanisms.CCR7 and ITGAE (CD103) are expressed on all mature DC types and on T cell subsets. BATF3 and ZBTB46 are shared with cDC2, and FLT3 with cDC2, pDCs and hematopoietic progenitors. IRF8 is highly expressed in pDCs and certain types of monocytes or macrophages. THBD (CD141/BDCA3) can be expressed on cDC2, pDCs, MoDCs and non-immune cell types. One study undertook the “tour de force” of profiling by microbulk RNAseq all the distinct mononuclear phagocyte types that they could identify in, and isolate from, BRCA or TNBC, in order to generate the cell-type specific transcriptomic signatures the most relevant to the cancer types studied (XCL1 and XCL2 express neither of these cell surface markers (A first issue that clearly stands out in  studied . However studied  encompas studied . Indeed, markers . More geThere is a need to define better transcriptomic signatures for human immune cell types, allowing to more rigorously computationally deconvolute the extent of their infiltration in tumors and its eventual correlation with the clinical outcome. One strategy to achieve this aim is to select genes which show high selective expression in the targeted immune cell type across tissues and activation conditions, as well as between human and mouse , 45, 153A second issue is the necessity to include signatures of various types of immune cells, to ensure that the better prognostic value observed in the patients whose tumors harbor higher levels of the genes specific to the candidate immune cell type is not merely a reflect of a higher overall leukocyte infiltration. Indeed, the goal is not just to compare globally “Hot” or “Warm” vs. “Cold” tumors. Rather, it is to pinpoint which immune cell types specifically promote tumor control, or on the contrary contribute to local immunosuppression, in order to identify how to best manipulate the tumor infiltrate, for the benefits of cancer patients, through combined immunotherapies. Thus, the cell types considered to be functionally and/or developmentally the most closely related to the candidate one should be included, for example cDC2 or MoDCs for cDC1, NK cells and γδ T cells for αβ T cells. In addition, one should also include cell types expected to have no, or opposite, impact on tumor growth, for example neutrophils, macrophages and regulatory T cells which are considered as promoting immunosuppression.FLT3L, XCL1, XCL2, CCL4, CCL5, LAMP3, CCR7, CXCL9, CXCL10, and CXCL11 (+ T cells for promoting effective antitumor immunity (A third issue is to deepen our understanding of when, where and how cDC1 promote tumor control. In several studies, the inferred higher cDC1 infiltration in tumors was correlated with higher inferred infiltrations of CTLs or NK cells, and with higher expression of d CXCL11  (55, 57,d CXCL11 , 80, 93.immunity . Finallyimmunity .A few clinical trials have already been conducted using treatment protocols that have been proposed to preferentially target/harness human cDC1  186, 18, 190. ThBuilding on the conservation of cDC1 molecular makeup and functions between mouse and human, similar tools have been generated in both species to specifically target these cells for immunotherapy against cancer. This should accelerate translation from mouse preclinical studies to human clinical trials. Hence, most of the tools and approaches that have been detailed in the section on mouse experimental models , 4 couldex vivo isolation, we and others recently developed optimized in vitro culture systems to generate high numbers of cDC1, cDC2 and pDCs from CD34+ hematopoietic progenitors (in vitro models will allow rigorous comparison of the functions of the different human DC types, dissection of their molecular regulation, and better understanding of their cross talk. Further adaptations of these protocols are warranted to derive in vitro autologous cDC1 from the circulating CD34+ cells of patients, load them with Ag and mature them, under conditions compatible for clinical use in vaccination or immunotherapy. It should be noted that encouraging results have been obtained with clinical trials of autologous ACT of ex vivo loaded and matured pDCs and cDC2 in melanoma patients, which seem superior to MoDCs to prime or boost endogenous CTL responses against the tumor. This emphasizes that, as in mice, cDC1 are not the only DC type that could be successfully harnessed for combined immunotherapy in cancer patients (To overcome the roadblock of the rarity of human cDC1 and of their fragility upon genitors , 148. Thpatients , 192.Considering their conserved specific expression pattern on mouse and human cDC1, and the very encouraging results obtained in mouse preclinical models, the CLEC9A and XCR1 receptors are the best candidates for Ag, or Ag+adjuvant cargo, delivery to human cDC1, using recombinant ligands , 194 or Systemic injection of FLT3-L could promote cDC1 differentiation and survival . Local dA systematic analysis of immune factor checkpoint expression on human DC types is ongoing in order to investigate which ones could be reasonable candidates as components of combined immunotherapies targeting both CTLs and DCs .+ T cells, and sometimes neutrophils are also central in promoting protective antitumor immunity, whereas Treg or type 17 CD4+ T cells, monocytes and macrophages may rather play immunosuppressive roles. Further studies using models allowing conditional depletion of cDC1 will be critical in rigorously investigating whether cDC1 functions are instrumental at the time when immunotherapies are delivered. These studies will definitely settle the currently prevailing hypothesis that cDC1 functions, when specifically boosted, could provide great support to boost patient responses to currently used anticancer immunotherapies.Lately, cDC1 have been in the spotlight of many studies investigating in mice the immune mechanisms driving tumor rejection, spontaneously or upon immunotherapy. All these studies converge toward a hub role of cDC1 in providing the initial priming, or in sustaining the activation, of antitumor T and NK cell responses. These advancements in our understanding of the role of cDC1 in antitumor immunity have been made possible by the recent blossoming of genetic tools allowing cDC1 manipulation. However, so far, most conclusions have been drawn from results obtained under experimental conditions that were not solely targeting cDC1, whether it was the use of genetically engineered mouse models or of mAb directing against cell surface markers. In fact, to be protective against immunosuppressive tumors such as those treated in the clinic, the immune response is necessarily complex and multi-parametric. More and more observations pinpoint that, in addition to cDC1, other DCs, type 1 CD4In human tumors, enrichment of genetic signatures described as cDC1-specific is associated with a good prognosis and a better clinical outcome in a several cancers, including luminal and TN breast cancer. These correlative analyses should be extended to additional types of cancer and to different patient treatment regimen. It is possible that the extent of cDC1 infiltration in the tumor fluctuates over time following the development or the suppression of an efficient antitumor immune response, as observed in mice during immunotherapy , and thaAll authors wrote the manuscript, contributed to manuscript revision, read and approved the submitted version.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.