While the fraction of Ki-67 positive cells, a snapshot marker for recent cell division, was higher in memory T-cells from blood compared to BM and LN, in vivo deuterium labeling revealed no substantial differences in the expected lifespans of memory T-cells between these compartments. Our results support the view that the majority of memory T-cells in the BM are self-renewing as fast as those in the periphery, and are continuously recirculating between the blood, BM, and LN.Memory T-cells are essential to maintain long-term immunological memory. It is widely thought that the bone marrow (BM) plays an important role in the long-term maintenance of memory T-cells. There is controversy however on the longevity and recirculating kinetics of BM memory T-cells. While some have proposed that the BM is a reservoir for long-lived, non-circulating memory T-cells, it has also been suggested to be the preferential site for memory T-cell self-renewal. In this study, we used  Immunological memory, the ability of the immune system to respond more quickly and strongly upon repeated antigen exposure, is the hallmark of the adaptive immune system. T-cell memory generated after the first antigen encounter can last for decades, and provides long-lasting immune protection , 2. It hPhysiological T-cell niches are an important factor in the maintenance of T-cell memory , 16. Theex vivo cell manipulation, which may interfere with cell homeostasis. A static marker like Ki-67 describes the division status of a cell at a given moment and location, but provides no information about cellular lifespans, and does not take into account that a cell may have proliferated previously, or elsewhere. In human studies, only static markers have been used to assess memory T-cell proliferation in organs other than blood  and carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling , 25, 27,an blood . Another+ and CD8+ T-cells in BM, blood, and lymphoid organs using in vivo stable isotope labeling, the state of the art technique to study lymphocyte dynamics in vivo. One of the great advantages of this technique is that the turnover of a cell population is traced regardless of time and space, allowing us to reliably follow the division history of a population. In addition, in vivo deuterium labeling is non-toxic and does not require ex vivo cell manipulation, enabling the study of an unperturbed system. To simultaneously quantify the lifespans of memory CD4+ and CD8+ T-cells in blood, BM and lymphoid organs we made use of the goat as animal model, taking advantage of its relatively large size to obtain enough T-lymphocytes from paired samples of blood, BM, and LNs.In this study, we simultaneously quantified the dynamics of memory CD4N = 34) were purchased from commercial farms and housed at Wageningen Bioveterinary Research, Lelystad, The Netherlands. Additional one-off surplus material from single blood samples taken for mandatory routine diagnostic tests were obtained from 8 adult female goats housed at the Department of Farm Animal Health, Faculty of Veterinary Medicine of the Utrecht University were used for IFN-ɤ ELISA assay.Female adult goats .2H2O)  was diluted to 4% in drinking water and administered ad libitum for 28 days. To determine deuterium enrichment in the body water, heparin plasma was collected during the up- and down-labeling phase, and was frozen and stored at −20°C until analysis.Deuterated water  at 17 different time points after start of label administration. During necropsy, the left and right pre-scapular LNs and the middle part of the sternum were isolated. Venous blood was collected from the jugular vein in heparinized Vacutainer (BD Biosciences) tubes prior to injection with pentobarbital. Single cell suspensions from LN were obtained by mechanical disruption, and from BM by flushing the sternum. BM cell suspensions were lysed with lysis buffer . Peripheral blood mononuclear cells (PBMCs) were isolated from blood using SepMate-50 tubes (Stemcell Technologies) and Ficoll-Paque Premium  following the manufacturer's protocol. The SepMAte-50 tubes were centrifuged at 1,400 g for 20 min. PBMCs were collected, spun down, and washed prior to cell staining and sorting.+CD8+) thymocytes were used to determine the positive gate for Ki-67, since double positive thymocytes have a clear population of cycling cells  and CD62L−CCR7−  CD4+ and CD8+ T-cells were sorted for transcriptome analysis  and CCR7− (memory) CD4+ and CD8+ lymphocytes were sorted from blood, BM and LN , CD62L−CCR7− (DN-M), CD62L+CCR7−, CD62L−CCR7+, total CCR7− (memory) and total CCR7+ (naive) CD4+ and CD8+ T-cells were sorted for functional assays.BM and LN cell suspensions and PBMCs were stained for extracellular markers using CD4-AF647 , CD8-PE , CD62L  conjugated with pacific blue (PB) , CCR7-PeCy7 , and CD14-Viogreen  monoclonal antibodies. For intracellular markers, cells were subsequently fixed, permeabilized , and stained intracellularly with Ki-67-FITC monoclonal antibody . Washing steps for intracellular staining were performed using Perm/Wash buffer (BD Biosciences). Double positive (CD4−CCR7− (DN-M), CD62L+CCR7+ (DP-N) CD4+, and CD8+ T-cells from blood and CD62L−CCR7− (DN-M) CD8+ T-cells from BM were sorted, spun down, and stored at −80°C prior to RNA extraction. Before thawing, cells were immersed in QIAzol Lysis Reagent (Qiagen). RNA was isolated and purified using the RNeasy kit (Qiagen). The concentration was measured on a NanoDrop ND-2000 (Thermo Scientific) and RNA integrity was examined using the 2200 TapeStation System with Agilent RNA ScreenTapes (Agilent Technologies).For RNA isolation, FACS sorted CD62LCapra hircus) genome GenBank assembly GCF_001704415.1_ARS1_rna transcripts  NCBI repository. Hybridization, washing, and scanning were performed according to manufacturer's instructions with an Agilent G2565CA scanner (Agilent Technologies).Total RNA (50 ng) combined with Spike A was used for amplification and labeling according to the Two-Color Microarray-Based Gene Expression Analysis guide using the Low Input Quick Amp Labeling Kit (Agilent Technologies). For the common reference an equimolar pool of all samples was made and amplified similarly as the test samples with the exception that Spike B was used. Synthesized aRNA was purified with the E.Z.N.A. MicroElute RNA Clean Up Kit (OMEGA bio-tek). The yields of aRNA and CyDye incorporation were measured on the NanoDrop ND-2000. An Agilent microarray (8 × 60 k) was custom-designed using the Agilent e-array microarray design tool v.7.6. The array contains 2,726 negative control probes, 1,319 Agilent control probes and 47,151 probes designed on transcripts from the goat  using the Limma package of Bioconductor. The “Normexp” method with offset = 16, was used for background correction and the resulting data was Quantile Normalized. Empirical Bayes statistics with Benjamini-Hochberg (BH) false discovery rate (FDR) correction was used to obtain statistical output for all to all comparisons.+CCR7+ (DP-N), CD62L−CCR7− (DN-M), CD62L+CCR7−, CD62L−CCR7+, CCR7− (memory), and CCR7+ (naive) CD4+ and CD8+ T-cells from blood and LN were cultured and stimulated with PMA (20 ng/ml) and ionomycin (1 ng/ml) for 70 h. Supernatant was collected at 20 and 70 h after stimulation, IFN-y production was measured using the BOVIGAM TB IFN-γ ELISA kit (Bovigam). Samples were tested in triplicates. IFN-γ optical density (OD) from unstimulated samples (background) was subtracted from the OD of stimulated samples.Sorted CD62L+CCR7+ (naive), CD4+CCR7− (memory), CD8+CCR7+ (naive), and CD8+CCR7− (memory) T-cells sorted from blood, LN and BM, and granulocytes using the ReliaPrep Blood gDNA Miniprep System  and stored at −20°C before processing for gas chromatography/mass spectrometry (GC/MS).Genomic DNA was isolated from CD42H2, M = 26) as previously described  (Deuterium enrichment in plasma and DNA was measured by GC/MS using an Agilent 5973/6890 GC/MS system (Agilent Technologies). Plasma was derivatized to acetylene (Cescribed . The derM = 435) . The der2H in body water over the course of the experiment, a simple label enrichment/decay curve was fitted to 2H enrichment in plasma:S(t) represents the fraction of 2H2O in plasma at time t (in days), f is the fraction of 2H2O in the drinking water, labeling was stopped at t = τ days, and δ represents the turnover rate of body water per day. The best fit for S(t) was used in the labeling equations for the different cell populations (see below). Up- and down-labeling of the granulocyte population was analyzed as previously described , we considered a steady state for each kinetic sub-population , and label enrichment of adenosine in the DNA of each sub-population i was modeled by the following differential equation:li is the total amount of labeled adenosine deoxyribose (dR) in the DNA of sub-population i and A is the total amount of adenosine in the cell population under investigation, c is an amplification factor that needs to be introduced because the adenosine dR moiety contains multiple hydrogen atoms that can be replaced by deuterium  in the DNA of cells that replicate at rate pi, and labeled adenosine is lost when cells of sub-population i are lost at rate pi. For naive T-cells this replication may occur both in the periphery and in the thymus. Scaling this equation by the total amount of adenosine in the DNA of sub-population i, i.e., defining Li = li/(αiA), yieldsLi represents the fraction of labeled adenosine dR moieties in the DNA of sub-population i. The corresponding analytical solutions aret ≤ τ), andt > τ).A mathematical model that allowed for kinetic heterogeneity between cells of the same population was fitted to the labeling data of the different leukocyte subsets. Each kinetic sub-population euterium , and pi L(t) = ∑αiLi(t), and the average turnover rate p was calculated from p = ∑αipi. Average lifespans were calculated as 1/p.The fraction of labeled DNA in the total T-cell population under investigation was subsequently derived from + CCR7− (memory) T-cells in blood, LN, and BM as well as CD8+ CCR7− (memory) T-cells in blood were significantly better described by a model including two kinetically different subpopulations while the other populations required only one.Because all enrichment data were expressed as fractions, labeling data were arcsin(sqrt) transformed before the mathematical model was fitted to the data. We followed a stepwise selection procedure to determine the number of kinetically different subpopulations to include in the model, adding a new kinetically different subpopulation into the model until the average turnover rate no longer significantly changed . For popp-value < 0.05 were considered significant.Differences between groups were assessed using Wilcoxon signed-rank test . Deuterium-enrichment data were fitted with the function nlm in R. The 95% confidence intervals were determined using a bootstrap method where the residuals to the optimal fit were resampled 500 times. Differences with a in vivo dynamics of memory T-cells simultaneously in blood, BM and LN, we made use of adult female goats as animal model. Immunophenotypic analysis showed that, in goats, BM lymphocytes include on average 1.4% CD4+ and 6.7% CD8+ T-cells, much lower percentages than in blood (18.2% CD4+ and 24.4% CD8+) and LN (31.9% CD4+ and 16.4% CD8+). BM also presented a lower CD4/CD8 ratio compared to blood and LN  and CC chemokine receptor 7 (CCR7), molecules that facilitate T cell homing to lymphoid tissues, than T-cells from blood and LN, with the majority of BM T-cells being CCR7−CD62L− , cell subset likely enriched for memory T-cells, and CCR7+CD62L+ , cell subset likely enriched for naive T-cells, T-cells. Transcriptome analysis on DN-M and DP-N CD4+ T-cells from blood and CD8+ T-cells from blood and BM confirmed at transcriptional level the expression of CD4, CD8A, CD8B, CCR7, and CD62L, and showed high expression of CD3E in all the samples despite the fact that CD4+ and CD8+ T-cells were not sorted based on CD3, because anti CD3 antibody is not available for goat  < 0.05], corresponding to 0.5% of the total  in DN-M compared to DP-N CD8+ T-cells included genes promoting T-cell survival and homeostasis, including TNFS1B and IL12RB2, molecules involved in immune activation, such as CD58, and genes involved in the cytotoxic effector function of T-cells, like GZMA and CD244 . We found that 70 h after stimulation, CD4+ and CD8+ DN-M T-cells were able to produce higher amounts of IFN-γ than their DP-N counterparts. In addition, DN-M T-cells reacted faster to stimulation, as a substantial amount of IFN-γ production was already detected at 20 h  T-cells are memory T-cells. In fact, we found the production of IFN-γ to be reliant on the expression of CCR7, not on CD62L, as CCR7−CD62L+ T-cells produced similar amounts of IFN-γ as CCR7−CD62L− and CCR7− T-cells, and higher amounts than CCR7+CD62L+ and CCR7+ T-cells .Finally, to h Figure . Altoget+ and CD8+ memory (CCR7−) T-cells. Ki-67 is a nuclear protein expressed during all phases of the cell cycle except for G0, thus actively dividing and recently divided cells express high levels of Ki-67. The percentage of Ki-67+ cells was significantly lower in memory T-cells from BM and LN compared to those in blood , with an average fraction of Ki-67+ cells of 3.2% of CD4+ and 3.9% of CD8+ memory T-cells from blood, 1.1% of CD4+ and 1.3% of CD8+ memory T-cells from BM, and 1.1% of CD4+ and 1.3% of CD8+ memory T-cells from LN  of the different cell populations, i.e., the fraction of cells replaced by new cells per day, and deduced the corresponding average lifespan (1/p) of the cells in that populations .Low percentages of Ki-67 positive cells in memory T-cells from BM have been interpreted as a sign that BM is the place where long-lived memory T-cells reside . However+ and CD8+ memory (CCR7−) T-cells from blood, BM, and LN were very similar  for CD8+ cells. Memory T-cells obtained from blood had an estimated average lifespan of 44 days  for CD4+ and 32 days  for CD8+ , and CD8+ T-cells 136 days   Figure . The lab+ and CD8+ T-cells highly correlated between BM and blood, as well as between LN and blood (both R2 > 0.70) and all data points were close to the x = y line  compared to naive (CCR7+) T-cells, in line with the typical observation in mice and humans that memory T-cells express higher levels of Ki-67 and reach higher deuterium enrichment than naive T-cells , accession number MB-P, MV, AK, JD, KT, and JB wrote the manuscript. MB-P, LR, AK, KT, and JB designed the experiments. MB-P, MV, LR, and AK performed the experiments. MB-P and MV analyzed the data. JD, RdB, and JB performed mathematical modeling.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.


Nectandra megapotamica (Spreng.) chemotypes and the balance of different classes of metabolites to verify how significant differences in plant metabolism are regarding chemotypes. We collected N. megapotamica leaves from eight adult plants in two Brazilian states. The essential oils and ethanol/water extracts were analyzed by GC-MS and LC-DAD-MS, respectively. Histochemical tests were performed, as well as chemical analyses of leaves from adaxial and abaxial foliar surfaces of N. megapotamica, and the stereochemistry of α-bisabolol was determined. Two different chemotypes, based on volatile compounds, were identified, distinguished by the presence of isospathulenol, α-bisabolol, β-bisabolene, and (E)-nerolidol for chemotype A, and bicyclogermacrene and elemicin for chemotype B. A stereochemical analysis of chemotype A extract revealed (+)-α-bisabolol enantiomer. Histochemical tests of chemotypes showed similar results and suggested the presence of essential oil in idioblasts stained with the dye NADI. The analyses of chemotype A leaves by GC-MS revealed similar compositions for abaxial and adaxial surfaces, such pattern was also observed for chemotype B. Medium and high polarity metabolites showed high chemical similarities between the chemotypes, highlighting the presence of proanthocyanidins and glycosylated flavonoids (O- and C-glycosides). Thus, N. megapotamica produced distinct volatile chemotypes with highly conserved medium to high polarity compounds. Such results suggest that phenolic derivatives have a basal physiological function, while genetic or environmental differences lead to differentiation in volatile profiles of N. megapotamica.Plants produce a wide range of secondary metabolites. Within a single species, chemotypes can be distinguished by the differences in the composition of the secondary metabolites. Herein, we evaluated  Plants can produce a variety of compounds, with different structural arrangements , and, baMurraya koenigii (L.) Spreng. (Rutaceae), also known as curry tree, presented two chemotypes, chemotype A, rich in pinene and sabinene, and chemotype B, which does not accumulate sabinene. The authors noted that sabinene contributes to aroma and flavor and highlighted that pinene and sabinene are valuable to the flavor industry, therefore, chemotype A is ideal for commercialization - as the principal ion fragment produced by the elimination of the glycoside (deoxyhexose and/or hexoside). In addition, the contraction of the ring followed by losses of CO (m/z 271 [Y0-H-CO]-) and a water molecule (m/z 255 [Y0-H-CO-H2O]-) [O-hexoside (peaks 10 and 13), quercetin O-deoxyhexoside , quercetin O-deoxyhexosyl-hexoside (peak 11). The compound 16 showed a loss of 132 u corresponding to a pentose and UV characteristic of flavonol (λmax ≈ 270 and 352 nm), thus, was identified as quercetin O-pentoside [The peaks entoside .14, 15, 18, 21, 22, and 23 were derived from luteolin and exhibit UV spectra characteristic of flavones (λmax ≈ 280 and 340 nm). The ion at m/z 285 was observed and yielded from losses of 308 u (deoxyhexose + hexose) for peaks 14, 15, and 18, characterizing these compounds as luteolin O-deoxyhexosyl hexoside [23 and 22 showed losses of 162 u (hexoside) and 132 u, suggesting the compounds luteolin O-hexoside and luteolin O-pentoside, respectively [21 was characterized as apigenin O-deoxyhexosyl hexoside because its fragmentation showed the fragment ion at m/z 269 (aglycone) yielded from the loss of 308 u (deoxyhexose + hexose).Peaks hexoside . The peaectively , 39. TheN. megapotamica was described by Garcez et al. [Nectandra genus, epicatechin was reported for N. amazonum [N. cissiflora [Nectandra cuspidata [O-rhamnoside in Nectandra grandiflora [Nectandra genus identified herein  are described for the first time, demonstrating the capacity of N. megapotamica to accumulate other O-glycosylated flavones and flavonols, as well as proanthocyanidins.The only previous report of phenolic compounds from z et al. , who desamazonum , N. cissssiflora , vitexinuspidata  and querndiflora . Thus, tN. megapotamica produced different volatile chemotypes. These compounds may remain unchanged due to basal actions that are exerted, acting as fundamental compounds for protection against ultraviolet, antimicrobial activity, response to biotic and abiotic stress, and protection against oxidative stress [In the present study, we observed that the secondary metabolism of medium to high polarity was highly conserved, even though e stress , 46. TheCryptocarya mandioccana Meisner, which presented a similar chemical profile for flavonoid glycosides, while the styrylpyrones presented high variability and were classified into four chemotypes. The authors attributed the production of such chemotypes to genetic or environmental influences (mainly soil and climate) [A similar study was done using another Lauraceae species, climate) .N. megapotamica has not yet been described. Such stereochemistry is important since there are high contents of bisabolol in leaves of N. megapotamica with chemotype A , and could be an important source of this compound for commercial use.α-Bisabolol is a monocyclic sesquiterpene that has two chiral centers (C1 and C7), and is found in nature in the following four isomers: (+) and (-)-α-bisabolol and (+) and (-)-epi-α-bisabolol -α-bisabolol by chiral column -α-bisabolol -α-bisabolol. Therefore, we conclude that α-bisabolol present in N. megapotamica is the compound (+)-α-bisabolol, since it presented a different retention time from (-)-α-bisabolol in a chiral column.Similarly, the samples were also analyzed by a chiral column. We detected an evident change in retention time comparing the α-bisabolol from The methods used for stereochemical identification are based on previously described analyses, such as Son et al  and AttiN. megapotamica from the S region has been reported [The presence of α-bisabolol in reported , howeverArtemisa annua [Cymbopogon flexuosus [Populus balsamifera [Populus balsamifera, which is rich in (+)-α-bisabolol, against rodents (Lepus americanus) [The (+)-α-bisabolol has been reported in the extracts of sa annua , Cymbopolexuosus , and Popsamifera . Some bisamifera . The comsamifera . In addiricanus) .N. megapotamica is a rich source of (+)-α-bisabolol with production up to 93.7% -α-bisabolol shows that S1 Table(PDF)Click here for additional data file.S2 TableNectandra megapotamica. S6A: analysis of adaxial surface; S6B: analysis of abaxial surface; S6IP: analysis of intact leaves; S7A: analysis of adaxial surface; S7B: analysis of abaxial surface; S7IP: analysis of intact leaves; n: number; RI: retention index of compound; RIL: retention index of literature.The individuals S6 and S7 were identified as (PDF)Click here for additional data file.S1 FigClick here for additional data file.S2 Fig.(PDF)Click here for additional data file.S3 Fig.(PDF)Click here for additional data file.S4 FigSample S6: Chemotype A; Sample C1 and C3: Chemotype B.(PDF)Click here for additional data file.S5 Fig.(PDF)Click here for additional data file.S6 Fig(PDF)Click here for additional data file.


Candida tropicalis YHJ1, and analyzed for application in xylitol production. The isolated yeast showed stress tolerance toward various chemical reagents and could grow with up to 600 g/L xylose in the culture medium. This yeast also had a broad carbohydrate utilization spectrum, and its xylitol yield was greatest in medium supplemented with xylose as the sole carbon source. In batch fermentation for xylitol production, the yeast could convert xylose prepared from acidic EPFBF pretreatment wastewater into xylitol. Interestingly, C. tropicalis YHJ1 xylose reductase, containing a Ser279 residue, exhibited more effective xylitol conversion compared to orthologous Candida enzymes containing Leu279 or Asn279; this improvement was associated with NADPH binding, as predicted through homologous structure modeling and enzyme kinetic analysis. Taken together, these results show a novel stress-tolerant yeast strain that may be applicable to xylitol production from toxic lignocellulosic byproducts.A stress-tolerant yeast was isolated from honey using acid hydrolysate generated from sequential acid-/alkali-pretreatment of empty palm fruit bunch fiber (EPFBF). The isolated yeast was identified molecularly, taxonomically, and morphologically as  Lignocellulosic biomass is a renewable resource for production of biofuels and chemical monomers through the biorefinery process  , the first-step enzyme in xylose metabolism, was also characterized in the isolated yeast and compared with recombinant mutant enzymes through computational homologous modeling for substrate binding.Isolation of a stress-tolerant strain rather than an adapted strain is an alternative approach to obtaining a potential microorganism for converting xylose in acid wastewater to xylitol. In this study, we isolated a stress-tolerant yeast from a honey jar using acidic hemicellulose extracts generated through sequential acid-/alkali-pretreatment of EPFBFs. A strain with resistance against various chemical stress reagents was identified molecularly and taxonomically as The EPFBF hydrolysate was prepared from the acid extract fraction for pretreatment through the sequential acid-/alkali-pretreatment process  yeast nitrogen base, adjusted to pH 6.0; then, the culture was incubated at 30°C with 240 rpm shaking for 120 h. After the first round of cultivation, 1/100 of the culture volume was inoculated into fresh medium, and then cultivated under the conditions described above for 72 h. This enrichment procedure was repeated five times. After enrichment, diluted culture was spread on an agar plate supplemented with the same medium composition used for the minimal medium, and the plate was then incubated at 30°C. Single colonies were isolated from the plates and cultivated in YPD broth . Cells were harvested from the pure culture, resuspended in 30% (v/v) glycerol (v/v), and maintained at −80°C for further strain identification.TM Genomic DNA Extraction Kit . The D1/D2 domain of the 26S rDNA gene and the internal transcribed spacer (ITS) region of the rDNA were amplified via polymerase chain reaction (PCR) with Phusion® high-fidelity DNA polymerase  and appropriate primer pairs, using the isolated strain's genomic DNA as the template. For PCR amplification of rDNA locus genes from eukaryotic strains, the primers used in this study were as follows: NL-1 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL-4 (5′-GGTCCGTGTTTCAAGACGG-3′) for 26S rDNA and ITS-1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS-4 (5′-TCCTCCGCTTATTGATATGC-3′) , an indicator plate that incorporates a chromogenic agent. The positive control C. tropicalis CBS94 and negative controls Candida albicans MYA-682 and MYA-2876 were also streaked onto the indicator plate for comparison of colony colors.To confirm the isolated strain as C. tropicalis strain, scanning electron microscopy (SEM) analysis was performed at the Korea Research Institute of Bioscience and Biotechnology  using a Hitachi S4300N . The freshly cultured strain was fixed in 0.1 M phosphate buffer (pH 7.2) containing 2.5% (v/v) paraformaldehyde-glutaraldehyde for 2 h, and then post-fixed in the same buffer containing 1% (w/v) osmium tetroxide for 1 h. Fixed samples were dehydrated in ethanol, substituted in isoamyl acetate, and then critical point-dried in CO2. The prepared samples were sputtered with gold using a Polaron sputter-coater (SC502) and analyzed via SEM.To observe the morphology of the isolated The isolated yeast was cultivated in YPD medium for 48 h at 30°C until the early exponential phase. Fatty acids were extracted, methylated, and separated using protocols of the MIDI/Hewlett Packard Microbial Identification System. Chemotaxonomic analysis based on the fatty acid composition of the cells was performed by the Korean Culture Center of Microorganisms .2O2; 0.25 mM or 0.5 mM calcofluor white; 15, 20, or 25 mM caffeine; and 100 mM xylose. Plates were incubated at 30°C for 3 days. To determine temperature resistance, spotted YPD plates were incubated at 25, 30, 37, 42, and 45°C.The isolated yeast strain was cultivated in YPD broth at 30°C. The culture was subsequently diluted to an optical density at 600 nm (OD600) of 0.1 and 4 additional 6-fold serial dilutions were performed. The cells were spotted on YPD agar plates. The conditions were: YPD with or without chemicals; 0.5, 1.0, or 3.0 M NaCl; 8, 10, or 16 % (v/v) ethanol; 3, 4, 5, or 10 mM HFor the carbohydrate utilization test, the yeast strain was freshly cultivated in YPD for 24 h, and then 1/1,000 of the culture volume was inoculated in minimal medium containing 2% (w/v) of the sole carbon source and 0.67 % (w/v) yeast nitrogen base without amino acids. The sole carbon sources used in this study include hexoses: D-fructose, D-glucose, D-galactose, D-mannose, and D-rhamnose; pentoses: L-arabinose, D-arabinose, D-xylose, and D-ribose; disaccharides: lactose, cellobiose, sucrose, maltose, melibiose, and trehalose; the trisaccharide D-raffinose; polysaccharides: starch, carboxymethylcellulose, avicel, xylan oat spelt, xylan birch wood, xylan beech wood, and inulin; sugar alcohols: glycerol, D-sorbitol, D-mannitol, and D-xylitol; sugar acids: D-gluconic acid, D-xylonic acid, D-glucuronic acid, and D-galactonic acid; alcohols: methanol and ethanol; and the amino sugar N-acetylglucosamine. Cells inoculated in each culture broth were cultivated at 30°C with 240 rpm shaking.For the xylitol production test supplemented with co-substrate, the yeast strain was cultivated in YPD, and then the culture was inoculated in basal medium (2% (w/v) xylose and 0.67% (w/v) yeast nitrogen base) supplemented with 2% (w/v) of an additional carbon source, D-glucose; D-galactose; D-mannose; D-fructose; D-xylose; D-sucrose; D-maltose; inulin; D-sorbitol; D-mannitol; D-trehalose; or D-raffinose. The inoculated culture broths were cultivated at 30°C with 240 rpm shaking.2PO4, 0.5% (NH4)2SO4, and 0.04% MgSO4·7H2O, adjusted to pH 6.0. The cells were cultivated at 30°C with shaking at 150 rpm.To test resistance against high concentrations of xylose for xylitol production, the yeast cell culture was inoculated in semi-defined medium composed of various concentrations of xylose , with 0.5% (w/v) yeast extract, 0.5% (w/v) KHCell growth and xylitol concentrations were monitored at 24-h intervals. The cell growth (OD600) was measured with a spectrophotometer based on the absorbance at 600 nm. The culture broth was centrifuged and the supernatant was analyzed for xylitol content through high-performance liquid chromatography (HPLC), as described below.C. tropicalis was cultivated in semi-defined medium containing 2% (w/v) xylose for 24 h, as described above. A 10% (v/v) seed culture of the yeast was inoculated into a 1-L fermenter FMT ST-S , with a 0.5-L working volume and basal medium containing 100 g/L (w/v) pure xylose or a solution of approximately 74 g/L xylose prepared from the biomass acid hydrolysate supplementing the basal medium. The fermenter was operated at 30°C with stirring at 150 rpm.For xylitol production, two xylose sources were used. One source was pure xylose chemical reagent, and the other was xylose solution prepared from the first dilute acid pretreatment of EPFBFs in the sequential acid-/alkali-pretreatment process -encoding xyl1, xylitol dehydrogenase-encoding xyl2, and xylulose kinase-encoding xyl3 genes were directly amplified from genomic DNA prepared using the protocol described above. Each gene was amplified through PCR with Phusion® high-fidelity DNA polymerase and the appropriate primer pairs using genomic DNA as the template. The primers used for gene amplification were as follows: xyl1-F (5′- ATGTCTACTACTCCTACTATTCCTAC-3′) and xyl1-R (5′-TTAAACAAAGATTGGAATGTTGTCCC-3′) for the xyl1 gene, xyl2-F (5′-ATGACTGCAAACCCATCATTAGTTC-3′) and xyl2-R (5′- CTATTCTGGACCATCAATTAAAC-3′) for the xyl2 gene, and xyl3-F (5′- ATGACTACTGATTATTCTGAAAACGAC-3′) and xyl3-R (5′- TTATTGTTTTAATAAAGTCTCTTCC−3′) for the xyl3 gene. The cloning procedures for the amplified genes followed the protocol described above for cloning of D1/D2 26S rDNA and ITS1-5.8S rDNA-ITS4 genes. The nucleotide sequences of the xyl1, the xyl2, and the xyl3 genes were deposited into the GenBank database under accession numbers MK690391, MK690392, and MK690393, respectively.To clone the gene family responsible for initial xylitol metabolism in C. tropicalis strains have yet been revealed, structure homology modeling was performed using the pseudo-quadratic restraints with simulated annealing (PQR-SA) method  (Protein Data Bank entry code: 1MI3) was used for the homologous protein models  .As no xylose reductase (XR) molecular structures from xyl1 gene fragment was amplified via PCR using the cloned vector described above and the appropriate primer set. The PCR products were digested with restriction enzymes, and the resulting DNA fragments were cloned into the expression vector pET39b to yield pET-XR. The plasmid contained an N-terminal 10xHis coding gene tag for purification. Finally, the recombinant vector was transformed into the E. coli strain BL21(DE3)pLysS.To produce recombinant XR, the XR-encoding xyl1-encoding gene, the plasmid pET-XR was used as a template. The oligonucleotide primers used for PCR in the forward direction were designed to generate S279L or S279N mutants. The primers for the reverse direction also contained mismatched bases for mutations (relevant bases are underlined): S279N, 5′-GAAACATTGCTGTTATTCCAAAATCAAACAATCCAGAAAGATTAGCTC-3′ (forward) and 5′- GAGCTAATCTTTCTGGATTGTTTGATTTTGGAATAACAGCAATGTTTC-3′ (reverse); S279L, 5′-GAAACATTGCTGTTATTCCAAAATCAAACTTCCAGAAAGATTAGCTCAAAAC-3′ (forward) and 5′-GTTTTGAGCTAATCTTTCTGGAEGTTTGATTTTGGAATAACAGCAATGTTTC-3′ (reverse). The primer melting temperature (Tm) and the optimum length of the primers were calculated using the Quick Change® primer design program from Stratagene . The 50-μL PCR reaction was carried out with 50–100 ng template, 0.2–2 μM primers, 200 μM dNTPs, and 2.5 U of Pfu Turbo DNA polymerase (Stratagene). The products of PCR amplification were treated with the restriction enzyme DpnI (Fermentas) for 2 h at 37°C. An aliquot of 5 μL of the PCR product described above was transformed into E. coli DH5α chemically competent cells and inoculated on a Luria-Bertani (LB) plate containing 30 mg ml−1 kanamycin. Several colonies were selected and their plasmids were isolated using plasmid mini-prep. All mutants were confirmed through DNA sequencing.To introduce the site-directed mutation into the E. coli strains harboring the recombinant plasmids were cultivated in Terrific Broth (TB) medium supplemented with 30 μg/mL kanamycin at 28°C for 24 h, and 0.5 mM IPTG was then added to the culture broth to induce lectin gene expression. After further cultivation at 25°C for 8 h, the cells were harvested, resuspended in lysis buffer , and then disrupted with three passes through a French press at > 1,000 psi. The crude lysate was centrifuged at 100,000 × g for 1 h at 4°C. Recombinant proteins from the clarified homogenate were purified through a three-step chromatography procedure using affinity chromatography HisTrap FF, ion-exchange chromatography HiTrap Q FF, and gel-filtration chromatography Superose 12 columns. Finally, the purified recombinant protein was analyzed using tricine-PAGE, pooled, and concentrated through ultrafiltration.To produce recombinant XR and its mutant enzymes, recombinant + per min using an extinction coefficient of 6.22 ×10−3 M−1·cm−1.Activities of the recombinant XR and its mutant enzymes were determined spectrophotometrically through monitoring changes in the absorbance at 340 nm, representing the oxidation of NAD(P)H. The enzymatic reaction was carried out at 30°C in 50 mM TrisHCl buffer (pH 6.8) containing 10 mM xylose, 10 mM NAD(P)H, 1 mM β-mercaptoethanol, and an appropriate concentration of the purified enzyme. Each activity determination was performed in triplicate in enzyme reaction mixtures obtained at different times. One unit of XR activity is defined as the amount of enzyme required to catalyze the formation of 1 μmol NADPLevels of xylose, other sugars, and organic acids were determined using an HPLC system equipped with a refractive index detector, an auto-sampler, and an Aminex HPX-87P column  for monosaccharide analysis, or a Rezex ROA-Organic Acid H+ column  for organic acid analysis, as described previously Kim, ,c, 2019.Honey, with its high sugar content, was a target resource for isolation of a stress-tolerant strain. Honey contains mainly carbohydrates, such as fructose, glucose, and other sugars, as well as organic acids including acetic, butanoic, formic, citric, succinic, lactic, malic, pyroglutamic and gluconic acids, and a number of aromatic acids. Additionally, honey contains hydroxymethylfurfural (HMF), a common product of hexose degradation below pH 5 , CBL Cd-3 (EU924133.1), LMICRO522 (KF746430), greater than that with other Candida species. Analysis of the ITS1-5.8S rDNA-ITS4 locus sequence and a phylogenetic tree constructed on the basis of these sequences revealed highly conserved relationships and tight clustering among C. tropicalis strains . This sequence also showed 99% identity with those of other Candida species, including C. tropicalis Ph43 (JN940623), C. albicans (GQ495089), C. parapsilosis (JN940627), C. tropicalis CBS94 (AY497695), C. maltosa (AY653546), and Lodderomyces elongisporus (AY653547)  . In cont strains . Analysi18:1 cis9 (ω9) (54.28%), C18:2 cis9 (9.79%), C16:0 (15.33%), and C16:1 cis9 (ω7) (11.57%). The minor fatty acids were C18:0 (5.37%), C17:0 (0.62%), C17:1 (1.57%), C15:0 (0.35%), C14:0 (0.86%), and C12:0 (0.27). The fatty acid composition was significantly different from those of other Candida stains  ethanol, 10 mM H2O2, and 15 mM caffeine. In addition, the strain showed a normal growth pattern in agar medium containing a high xylose content (100 g/L). Therefore, its growth was further evaluated under liquid culture conditions with a high xylose concentration. On the other hand, the strain was sensitive to high concentrations of calcofluor white, which might lead to inhibition of chitin and β-glucan synthesis for yeast cell wall formation , pentose sugars (xylose), disaccharides , trisaccharide (raffinose), sugar acids (gluconate and xylonate), sugar alcohols , and an amino sugar (N-acetylglucosamine) in minimal medium for cell growth  of C. tropicalis MYA-3404, 99% identity with the xylitol dehydrogenase (ABG49459.1) of C. tropicalis Ct1799, 99% identity with a hypothetical protein CTRG_03873 (XP_002549576.1) of C. tropicalis MYA-3404, and 80% identity with the D-xylulokinase (AAY87404.1) of C. maltose Xu316. The nucleotide sequences of the C. tropicalis YHJ1 xyl1, xyl2, and xyl3 genes were deposited in GenBank under accession numbers MK690391, MK690392, and MK690393, respectively.To identify the genes involved in initial xylose metabolism in C. tropicalis YHJ1 XR has 99% identity with counterpart proteins in other Candida strains, three variable residues in its sequence, Asn279Ser, Lys296Gln, and Asp297Glu, were observed through comparison with the C. tropicalis MYA-2404 XR sequence. To determine whether these variable residues could interact with or bind to xylose or a coenzyme acting as a co-substrate NADPH, structure homology modeling was performed. The protein crystal structure (PDB accession number 1MI3) of CtXR (green)  and C. tropicalis MYA-2404 XR (blue). Homology modeling with C. tenuis XR (Protein Data Bank entry code: 1MI3) revealed sequence identity of 0.78 for C. tropicalis YHJ1 XR and 0.76 for C. tropicalis MYA-2404 XR. Three superimposed structures of Candida XR proteins were constructed  of structed . These tproteins .C. tropicalis MYA-2404 XR may interact repulsively with the positively charged phosphate group, which might destabilize the binding of NADPH to the protein. In addition, the Leu277 residue in C. tenuis XR has a non-polar tendency, causing weaker binding of NADPH compared to the other residues in C. tropicalis XR structures. Thus, the Asn279 or Leu277 residue might fail to bind or interact with the non-phosphorylated cofactor NADH due to the long distance between the ligand and the side chain of the amino acid.On the other hand, the Asn279 residue in C. tropicalis YHJ1 XR affects catalytic activity, two mutants of the wild-type XR containing Ser279Asn or Ser279Leu were constructed. The his-tag fused recombinant wild-type C. tropicalis XR (XR_S279) and these two mutants (XR_S279L and XR_S279N) were produced under identical culture conditions, as described in the materials and methods section. Analysis through tricine-PAGE, as well as western blotting of the induced E. coli extract, showed dense bands of expressed protein at the expected position of approximately 34 kDa (To confirm that the Ser279 residue in y 34 kDa . The wilC. tropicalis YHJ1 strain has higher activity toward the phosphorylated cofactor NADPH, by approximately 1.4-fold, compared to XR_S279L and 1.5-fold for XR_S279N.The specific enzyme activities of the wild-type and mutant XR enzymes were determined using NADPH or NADH as a cofactor, and these data are summarized in In biorefinery processes using a renewable bioresource, biomass pretreatment is a key step for turning degradable biopolymers into fermentable sugars. Depending on the biomass materials employed, numerous approaches using physical, chemical, biological, or hybrid methods have been applied to optimize the treatment process by lowering cost and energy consumption (Melin and Hurme, C. tropicalis YHJ1, through phylogenetic, taxonomic, and morphological analysis (C. tropicalis YHJ1 showed a strongly a resistant phenotype to high concentrations of xylose up to 600 g/L, despite reduced cell growth dependent on the sugar content in the culture medium (C. tropicalis has been known as a virulence event for colonization and infection of different host niches (Homann et al., C. tropicalis strain under high concentration of xylose, the spotting assays showed calcofluor white, a chitin synthase inhibition, inhibited strongly the cell growth (In this study, we used neutralized acid EPFBF hydrolysate to isolate a stress-tolerant strain for application in xylose fermentation to produce sugar alcohol. Honey was used as a candidate resource for isolation of a stress-tolerant strain that could utilize xylose, a fermentable sugar found in the acidic wastewater from sequential acid-/alkali-pretreatment of biomass. Although honey is primarily made up of sugars, sugar acids, and HMF are also present as minor compounds (Zirbes et al., analysis . This yeanalysis . Interese medium . The rese medium . The crie medium . Interesl growth . These rC. tropicalis CB92 (C. tropicalis YHJ1 showed greater utilization capacity for xylitol than strain CB92. The isolated yeast might have more effective enzymes or transporters for xylitol metabolism. In addition to carbohydrate utilization, C. tropicalis YHJ1 produced the highest xylitol concentration in medium supplemented with only xylose, and no other carbohydrate co-substrates (In the carbohydrate utilization test using minimal medium supplemented with a single carbon source, the isolated strain showed similar utilization patterns for various hexose and pentose sugars, disaccharides, polysaccharides, and sugar alcohols to those of the counterpart stain lis CB92 . Howeverbstrates . Cells cbstrates .Candida strains (Lima et al., Batch fermentation using a xylose solution prepared with acid pretreatment wastewater from sequential acid-/alkali-pretreatment of EPFBFs showed a lower xylitol concentration and 40% lower yield compared to the culture supplemented with pure xylose . The xylCandida strains, a transported xylose is first reduced by NADPH- or NADH-dependent XR, encoded by the gene xyl1, to create xylitol. Xylitol, a sugar alcohol, is then oxidized by NADP+- or NAD+-dependent xylitol dehydrogenase, encoded by the gene xyl2, to form xylulose. Xylulose is further metabolized to xylulose-5-phosphate by ATP-dependent xylose kinase, encoded by the gene xyl3. Subsequently, this phosphate sugar enters the pentose-phosphate pathway. The deduced amino acid sequences of the xyl1, xyl2, and xyl3 genes involved in xylose metabolism in C. tropicalis YHJ1 had 99% identity to their counterpart sequences reported from other C. tropicalis strains. Nevertheless, XR, the enzyme mediating the first step of xylitol production, has a different amino acid residue in C. tropicalis YHJ1, which could influence the tight interaction between the cofactor NADPH and the active site of the protein, as shown through structure homology modeling (C. tropicalis YHJ1 XR might closely interact with the phosphate moiety of NADPH, but this would not occur with Leu279 or Asn279 in the putative cofactor binding pocket. Although the overall amino acid sequences of XRs do not significantly differ among Candida strains, minor sequence differences near enzyme active sites might affect the catalytic activities as well as the selectivity for a phosphate or non-phosphate cofactor. However, further enzymatic kinetics studies are needed to evaluate the catalytic activities of the wild-type XR and other mutants with other non-phosphorylated cofactors and compounds that are inhibitory to xylitol production.During the initial step of xylose metabolism in modeling . In addiC. tropicalis YHJ1 was isolated from honey and molecularly identified based on phylogenetic, taxonomic, and morphological analyses. The stress-tolerant yeast tolerated various chemical reagents and could grow with up to 600 g/L xylose for xylitol production. The yeast showed a broad carbohydrate utilization spectrum, with its greatest xylitol yield being in medium supplemented with xylose as the sole carbon source due to catabolic repression. Batch fermentation for xylitol production using xylose solution prepared from acidic EPFBF pretreatment wastewater showed lower xylitol yield compared to the culture supplemented with pure xylose. Nevertheless, XR containing the Ser279 residue in C. tropicalis YHJ1 allowed for more effective xylitol conversion compared to orthologous Candida enzymes containing Leu279 or Asn279. This residue might influence the tight interaction with the cofactor NADPH, as shown in the homologous protein simulation and enzymatic analysis. This study describes a novel stress-tolerant yeast strain and its XR, which can be used for xylitol production from a sugar solution based on biomass pretreatment wastewater.The xylose-fermenting yeast The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.SK conceived and designed the experiment and performed the experiment. SK and BS analyzed data. JL performed the computational homologous protein modeling. All authors have wrote the manuscript, read, and approved the final 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.


Mycobacterium tuberculosis antigen-specific CD4 T cells in a cohort of HIV-infected persons starting antiretroviral treatment (ART) in a high tuberculosis endemic area is described. Restoration of the antigen-specific CD4 T-cell subsets mirrored the overall CD4 T-cell compartment. Activation (assessed by HLA-DR expression) decreased during ART but remained elevated compared to HIV-uninfected persons. Despite known M. tuberculosis sensitization determined by interferon-γ release assay, 12/23 participants had no M. tuberculosis-specific CD4 T cells detectable by flow cytometry, combined with overall elevated T-cell activation and memory differentiation, suggesting heightened turnover. Our data suggest early ART initiation to maintain polyfunctional immune memory responses.The reconstitution of  Mycobacterium tuberculosis-specific T cells early during HIV-1 infection [M. tuberculosis-specific CD4 T-cell responses over the first 6 months of ART have not been studied in detail and data on the recovery dynamics of antigen-specific cellular immunity remain scarce. Here we compare the reconstitution of the whole CD4 T-cell compartment to that of the M. tuberculosis antigen-specific CD4 T-cell compartment, during the first 6 months of ART using flow cytometry, in a cohort of M. tuberculosis-sensitized HIV-infected persons starting ART, in an area with high prevalence of latent tuberculosis infection [Human immunodeficiency virus HIV-1) infection is the biggest risk factor for development of tuberculosis. Antiretroviral therapy (ART) reduces the risk of tuberculosis in HIV-1–coinfected persons. This therapy is associated with tuberculosis decline in sub-Saharan Africa between 2003 and 2016, with an estimated 1.88 million tuberculosis cases prevented [ infectionfection . Increasnfection . HoweverM. tuberculosis sensitization was determined by QuantiFERON Gold In-tube (QFT) assay and an in-house enzyme-linked immunospot assay (ELISPOT) as described and previously reported [M. tuberculosis sensitized as assessed by ELISPOT and QFT during the longitudinal follow-up [The University of Cape Town’s Faculty of Health Sciences Human Research Ethics Committee approved the study (REC: 245/2009) and written informed consent was obtained from all participants. Fifty HIV-1 infected persons were recruited at enrolment into the ART program from the Ubuntu Clinic in Khayelitsha, South Africa, as described [ollow-up . None ofM. tuberculosis whole-cell lysate  for a total of 16 hours. Brefeldin A  was added 3 hours after the onset of stimulation. After stimulation, cells were washed, stained with LIVE/DEAD Fixable Near-IR Stain (Invitrogen), and subsequently surface stained with the following antibodies: CD14-APC/Alexa Fluor 750 (Invitrogen) and CD19-APC/Alexa Fluor 750 (Invitrogen), CD4-FITC (Becton Dickinson), CD27-BV711 (Becton Dickinson), CD45RA-BV570 (Becton Dickinson), killer cell lectin-like receptor G1 (KLRG1)-PerCP/eFluor 710 (eBioscience), and HLA-DR–PE (Becton Dickinson). Cells were then fixed and permeabilized using Cytofix/Cytoperm buffer (Becton Dickinson) and stained with CD3-BV650 (Becton Dickinson), interleukin-2 (IL-2)–PE/Dazzle (Biolegend), tumor necrosis factor-α (TNF-α)–eFluor 450 (eBioscience), and interferon-γ (IFN-γ)–Alexa Fluor 700 (Becton Dickinson). Finally, cells were washed and fixed in 1% formaldehyde containing phosphate-buffered saline. Samples were acquired on a BD Fortessa flow cytometer and analyses were performed using FlowJo . A positive M. tuberculosis response was defined as at least twice the background . For cell surface phenotyping, only M. tuberculosis response with more than 20 events were considered. Cell polyfunctionality was analyzed using Pestle and Spice software. The gating strategy is presented in Cryopreserved PBMC were thawed and rested in RPMI 1640 containing 10% heat-inactivated fetal calf serum prior to antigen stimulation. PBMC were stimulated using P value <.05 was considered statistically significant.Statistical analyses were performed using GraphPad Prism (version 5.0). Nonparametric statistical tests were used for all comparisons. The Mann-Whitney U test and Wilcoxon signed-rank test were used for unmatched and paired samples, respectively, and the Kruskal-Wallis ANOVA using Dunn test for multiple comparisons. Correlations were performed using the nonparametric Spearman rank test. A M. tuberculosis-specific CD4 T cells in 23 HIV-infected individuals at baseline (day 0 of ART initiation) and at 1, 3, and 6 months of ART, in parallel with the reconstitution profile of the whole CD4 T-cell compartment, compared to a group of 28 HIV-uninfected individuals.We longitudinally analyzed the frequency and activation profile of 3). Thereafter, the increase in CD4 count was more modest, reaching medians of 299 and 331 cells/mm3 at 3 and 6 months, respectively. This is further illustrated by the fact that the median gain in CD4 T cells/month was significantly higher during the first month of ART (77 cells/month) compared to the following months (approximately 18 cells/month). These changes in absolute CD4 count coincided with successful suppression of HIV viral load, with the median plasma viral load becoming undetectable at 3 months post-ART . A normalization of the CD4 T-cell memory profile was observed at 6 months of ART, where the median proportion of naive cells significantly increased and late differentiated cells decreased compared to baseline . CD4 T-cell activation, as assessed by HLA-DR expression on CD4 T cells, progressively decreased after ART initiation . However, CD4 activation after 6 months of ART remained significantly elevated compared to HIV-1 uninfected persons . Moreover, they underwent a significantly lower gain in absolute CD4 count over 6 months of ART compared to M. tuberculosis responders   , suggestM. tuberculosis response, the frequencies of M. tuberculosis-specific CD4 T cells did not change significantly during the first 6 months of ART. However, due to CD4 T-cell reconstitution, this translates into a significant increase in the absolute number of M. tuberculosis-specific CD4 T cells as soon as 1 month post-ART initiation (M. tuberculosis-specific CD4 T cells did not change on ART (data not shown), as previously described [M. tuberculosis-specific CD4 T cells decreased, indicated by the significant fall  of HLA-DR expression on these cells from baseline to 6 months of ART. Notably, the activation profile of M. tuberculosis-specific CD4 T cells mirrored that of total CD4 T cells both at baseline and 6 months of ART. Additionally, the rate of cell “deactivation” after 6 months of ART was comparable in M. tuberculosis-specific cells and the overall CD4 compartment . It was interesting to note that individuals with undetectable M. tuberculosis responses exhibited elevated activation and increased memory differentiation in their CD4 compartment pre-ART initiation compared to participants with maintained M. tuberculosis-specific CD4 T-cell responses. It is thus possible that heightened cell turnover, fueled by HIV-induced systemic immune activation, could have contributed to the depletion of M. tuberculosis-specific memory CD4 responses to levels undetectable by flow cytometry [The study cohort originated from Khayelitsha township, a highly tuberculosis endemic area, with the prevalence of tely 69% . When asribed in , 12 out ytometry .M. tuberculosis-specific response pre-ART, the frequency of M. tuberculosis-specific CD4 T cells remained stable over time even with a significant expansion of the total number of circulating CD4 T cells. This indicates that the restoration of M. tuberculosis-specific CD4 T cells mirrors the reconstitution of the overall CD4 compartment. Similarly, the “deactivation” of M. tuberculosis-specific CD4 cells paralleled the change observed in the total CD4 T cells. The functional capacity of M. tuberculosis-specific CD4 T cells evolved towards a more polyfunctional profile in response to ART, whereby cells regained the ability to produce IL-2. A shift to dominance of CD4 T cells secreting both IFN-γ and IL-2 has been associated with M. tuberculosis clearance [M. tuberculosis protection. However, in individuals without detectable M. tuberculosis response, 6 months of ART did not result in detectable M. tuberculosis-specific response. The slower rate of CD4 reconstitution observed in these participants  may in part explain the lack of restoration of M. tuberculosis-specific CD4 T cells. As it has been described that M. tuberculosis-specific CD4 T cells are a preferential target for HIV [M. tuberculosis-specific memory CD4 T cells, predisposing these individuals to increased risk of active tuberculosis.During 6 months of ART, in participants with a detectable learance . It is tM. tuberculosis.While our study has a number of limitations, being a preliminary study with small numbers, the results overall support and reinforce the relevance of generalized recommendation of early ART initiation, to decrease the risk of tuberculosis in HIV-infected individuals, as it may contribute to the maintenance of polyfunctional CD4 T-cell based immune memory responses against The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at jiz417_suppl_Supplementary_FigureClick here for additional data file.


Background and Aims: Entecavir (ETV) resistance of hepatitis B virus (HBV) conventionally requires rt184, 202, or 250 mutations plus lamivudine-resistance mutation (rtM204V/I ± L180M). This study aimed to clarify whether rtL180M+A181C+M204V mutations may contribute to HBV ETV resistance.Methods: Serum samples were collected from 22,009 patients who underwent resistance testing in Beijing 302 Hospital from 2007 to 2016. HBV reverse transcriptase (RT) gene was screened by direct sequencing and verified by clonal sequencing. Phenotypic analysis was performed for evaluating replication capacity and drug susceptibility.Results: Classical ETV-resistance mutations of HBV were detected in 1252 patients who were receiving ETV therapy. The rtA181C mutation was detected with rtL180M+M204V mutations in 18 lamivudine-experienced ETV-treated patients, and the emergence of the mutations was associated with virological breakthrough or inadequate virological response to ETV. Patient-derived representative rtA181C-containing mutants, rtL180M+A181C+M204V, rtL180M+A181C+M204V+M250V, and rtL180M+A181C+S202G+M204V, exhibited 45.7%, 25.9%, and 25.0% replication capacity and 85.6-, 356.1-, and 307.1-fold decreased susceptibility to ETV respectively compared to the wild-type strain, while the three mutants remained sensitive to tenofovir (TDF). Artificial elimination of rtA181C largely restored the rtL180M+A181C+M204V mutant’s sensitivity to ETV. Molecular modelling of viral RT binding to ETV showed that the rtL180M+A181C+M204V mutant had a less stable conformation compared to rtL180M+M204V mutant. In clinical practice, undetectable serum HBV DNA was achieved in two of five longitudinally followed rtA181C-positive patients who received switching-to TDF therapy, but not in the other three who received add-on adefovir therapy during observation.Conclusions: Both clinical and experimental data support rtL180M+A181C+M204V as a novel non-classical ETV-resistance mutation pattern. It has been estimated that there are 240 million people with chronic HBV infection worldwide and around 78 million of them are in China ,2. ChronETV is a potent antiviral, which has a high barrier to resistance and is recommended as first-line anti-HBV agent. Signature or classical ETV-resistance mutations require an HBV RT mutation at position rtT184, S202, or M250 in the presence of LAM-resistance mutations rtM204V+L180M or rtM204I ± L180M (abbreviated as LAMr) . In addiIn recent years, we have identified several unusual HBV RT mutations associated with LAM-, ADV-, and multidrug-resistances based on genotypic analysis of a large number of NA-treated patients in combination with phenotypic analysis . In thisHBV ETV-resistance mutations were detected in 5.69%  of total patients, and in 20.29%  of ETV-experienced patients enrolled in the study. Among the 1252 patients, only 17 patients (1.36%) had no exposure history to other nucleotide analogues such as LAM and LdT. HBV genotypes B, C, and D  were determined for 143 (11.42%), 1096 (87.54%), and 13 (1.04%) patients respectively. Specifically, rtT184, rtS202, rtM250, rtT184+S202, rtT184+M250, and rtS202+M250 substitution-based mutation types accounted for 41.29%, 35.86%, 14.38%, 7.35%, 0.72%, and 0.40% of ETV-resistance mutations, respectively. The patterns of LAMr, in the composition of ETV-resistance mutations, included rtM204V+L180M (82.91%), rtM204I ±L180M (15.25%), and rtM204V /I+L180M (1.84%) . The nucThe rtA181C mutation was detected in 18 patients by direct sequence analysis, encompassing 0.08%  of the study population and 0.29% (18/6170) of ETV-experienced patients. All rtA181C-positive patients experienced various sequential/combined NAs therapies including ETV and had a history of LAM exposure prior to ETV treatment. The median duration for ETV therapy was 39 (7–96) months before rtA181C was detected. Coexistence of rtL180M+M204V mutations was detected in all rtA181C-positive patients. Clonal sequencing verified that rtL180M+A181C+M204V mutations truly coexisted in the same viral genomes for all 18 samples. In addition, the colocalization of rtL180M+A181C+M204V mutations and rtT184A or rtM250V mutation on the same viral genome was verified in the samples of three patients . The representative cloned rtL180M+A181C+M204V sequences from each patient, together with other rtA181C-containing sequences, have been deposited in GenBank (accession number: MF682469-MF682490). Among the 18 patients, 15 (83.33%) were infected with HBV/C and three (16.67%) with HBV/B. The HBV genotype distribution showed no significant difference when compared with that of the rtA181C-negative patients across the studied population . The clinical data and HBV mutation patterns, by direct and clonal sequencing, of the 18 patients are summarized in Serial serum samples were obtained from five rtA181C-positive patients  with a failure of sequence analysis of sample A3  in concomitance with rtL180M+A181C+M204V mutants , rtA181V mutants (8% in E3), rtL180M+A181C+M204V+M250V mutants (4% in E3), and rtL180M+A181C+S202G+M204V mutants (4% in E3)  were classified as genotype B, and the sequences from the other 13 patients were classified as genotype C (50 of ETV (representing a 70.5-fold ETV resistance) compared to the wild-type .Phenotypic analysis was performed for four mutants and one wild-type strain derived from serial serum samples of patient 5, as well as for two classical ETV-resistance mutants and one wild-type strain of genotype C HBV  derived from another ETV-refractory patient . In addivs. −4.97 kcal/mol). Additional introduction of rtS202G or rtM250V into rtL180M+A181C+M204V mutation pattern resulted in one N–H:O bond reduction with further increased binding energy . In addition, the root mean square fluctuation (RMSF) graph showed that the flexibility of the mutant RT was obviously reduced, indicating that rtL180M+A181C+M204V mutation led to greater protein rigidity and therefore made the HBV RT relatively unsuitable for interacting with ETV-TP .HBV has the potential to evolve under environmental pressure through the selection of adaptive mutations that the most fit mutants may emerge depending on their replication capacity, and their sensitivity to antivirals and to the host immune responses ,18. Drugin vitro  that covers the full-length RT region nt 130-1161) and overlapping S region nt 155-835). Clonal sequencing was performed using the TA cloning strategy, and drug-resistance mutations were analysed as described previously . This HBV RT modelling structure was used as a wild-type to build the structures of the HBV NA-resistant mutants. Autodock software  was used to simulate the docking process and evaluate the binding energy of HBV RT and ETV-TP. Autodock is a component of the MGLTools that is freely available for academic use. More detailed information about Autodock could be found at the following website: http://mgltools.scripps.edu/.The modelling structure of the HBV RT was constructed by SWISS-MODEL  with a cubic box water model . The root-test. Statistical analysis was carried out in the Statistical Program for Social Sciences . A P-value of <0.05 (two-tailed) was considered statistically significant.Data were presented as the mean ± standard deviation, or the median (range). Differences between variables were examined by Student’s


NL-G-F (APPKINL-G-F) mice. Substantial phosphorylation of IRS1 at specific Ser sites occurs in type 2 diabetes- or age-related memory deficits independently of amyloid-β (Aβ). Furthermore, we present the first evidence that, in APPKINL-G-F mice showing Aβ42 elevation, the increased phosphorylation of IRS1 at multiple Ser sites occurs without memory impairment. Our findings suggest that the phosphorylation of IRS1 at specific Ser sites is a potential marker of Aβ-unrelated memory deficits caused by type 2 diabetes and aging; however, in Aβ-related memory decline, the modifications of IRS1 may be a marker of early detection of Aβ42 elevation prior to the onset of memory decline in AD.The biological effects of insulin signaling are regulated by the phosphorylation of insulin receptor substrate 1 (IRS1) at serine (Ser) residues. In the brain, phosphorylation of IRS1 at specific Ser sites increases in patients with Alzheimer’s disease (AD) and its animal models. However, whether the activation of Ser sites on neural IRS1 is related to any type of memory decline remains unclear. Here, we show the modifications of IRS1 through its phosphorylation at etiology-specific Ser sites in various animal models of memory decline, such as diabetic, aged, and amyloid precursor protein (APP) knock-in  Insulin signaling mediated by insulin receptor substrates 1 and 2 (IRS1 and IRS2) is involved in the regulation of growth, glucose homeostasis, energy metabolism, and lifespan ,4. The bAmong the numerous Ser residues on IRS1, a few sites, including human(h)Ser312/mouse(m)Ser307, hSer616/mSer612, hSer636/mSer632, hSer639/mSer635, and hSer1101/mSer1097, have been studied because a limited number of phosphospecific antibodies were previously commercially available.Nonetheless, hSer312/mSer307 has been widely investigated and implicated in insulin resistance under metabolic stress conditions and in inflammatory conditions such as obesity, hyperinsulinemia, and dyslipidemia ,9,10. HoNL-G-F (APPKINL-G-F) mice, a novel AD mouse model, remains unclear [In the central nervous system, studies on postmortem brain tissues of patients with Alzheimer’s disease (AD) have revealed increased phosphorylation of IRS1 at hSer312/mSer307, hSer616/mSer612, hSer636/mSer632, and hSer639/mSer635 compared with that in non-AD control subjects ,14,15,16 unclear . Meanwhi unclear ,21,22. F unclear . NL-G-F mice and whether it occurs before or after the onset of memory decline in APPKINL-G-F mice. We demonstrate that the concomitant activation of specific Ser sites on hippocampal IRS1 with amyloid-β (Aβ)-unrelated memory decline occurs in DIO and aged mice, whereas STZ mice exhibit memory deficits independent of IRS1 activity. We further show that increased phosphorylation of hippocampal IRS1 at Ser sites is already observed in young APPKINL-G-F mice showing normal memory function despite increased Aβ42 level. These data suggest that the activation of Ser residues on hippocampal IRS1 is associated with non-AD-related memory impairment in T2DM and aging and with Aβ42 level, which is related to the onset of cognitive decline in AD. In the present study, we investigated whether the modification of hippocampal IRS1 by Ser phosphorylation is commonly associated with different types of memory decline in DIO, STZ, aged, and APPKINL-G-F (Swedish (NL), Arctic (G), and Beyreuther/Iberian (F) mutations) homozygous mice were obtained from Dr. Saido at the Laboratory for Proteolytic Neuroscience, RIKEN Brain Science Institute, Saitama, Japan [NL-G-F mice, and their respective age-matched control mice . All mice were housed in a standard 12 h light–dark cycle with free access to water and food (room temperature: 25 ± 2 °C). Animal experiments were performed in compliance with the guidelines and with the approval of the ethics committee in Animal Care and Use of the National Center for Geriatrics and Gerontology in Obu, Aichi, Japan .C57BL/6J male mice (4 weeks of age) supplied by Japan SLC, Inc.  were used to establish type 1 and 2 diabetes mellitus  mice and their respective control mice. Generation of high-fat-diet (HFD)-induced type 2 diabetes mice (DIO mice) was carried out as previously described . Brieflya, Japan . Age-matThe protocol of western blotting used has been previously described . In brieHippocampal-dependent spatial memory was tested using a water T maze ,25 accor® Aviva; Roche DC Japan K.K.). The level of plasma insulin at 6 h fasting was determined using an insulin enzyme-linked immunosorbent assay kit .Body weight and the blood glucose levels were recorded after 6 h of fasting. Blood glucose levels were measured using a portable glucose meter  containing a protease inhibitor cocktail  and a phosphatase inhibitor cocktail  with a pellet mixer. After incubation on ice for 15 min, the lysates were centrifuged at 14,200× g for 5 min at 4 °C and the supernatants were placed in a fresh tube. Protein concentration was determined using a Bicinchoninic acid (BCA) protein assay kit . The levels of T-PER-extractable Aβ were measured with the Human β Amyloid (1–40) enzyme-linked immunosorbent assay (ELISA) Kit Wako II , the Human β Amyloid (1–42) ELISA Kit Wako, High Sensitivity , the Human/Rat/Mouse β Amyloid (1–40) ELISA Kit Wako II , and the Human/Rat/Mouse β Amyloid (1–42) ELISA Kit Wako, High Sensitivity  in accordance with the manufacturers’ instructions.In order to measure the Aβ levels in the hippocampus, hippocampal tissue was isolated on ice and homogenized in a lysis buffer . Significance is indicated as * p < 0.05 and ** p < 0.01.All results are presented as mean ± standard error of the mean (SEM) in the text. Statistical analyses were performed using Prism7 for Mac OS X v.7.0d . Data were statistically analyzed using Student’s We recently reported that T2DM-related cognitive declines in 45-week-old DIO mice fed a 60% HFD for 42 weeks were accompanied by an increased phosphorylation of hippocampal IRS1 at mSer1097 . While mFurthermore, we examined whether memory impairment in middle-aged DIO mice is linked to increased levels of Aβ42, a pathological feature of AD. Biochemical analysis with specific kits  demonstrated that, compared with age-matched wild-type (WT) mice, there was no change in Aβ40 and Aβ42 levels in the T-PER fractions obtained from the hippocampi of 35-week-old DIO mice D.In contrast to DIO mice at 45 weeks of age, the phosphorylation of IRS1 at mSer307, which increases in insulin resistance, diabetes, and obesity ,28, the To investigate whether the alteration of IRS1 through Ser phosphorylation is associated with type 1 diabetes mellitus (T1DM)-induced memory deficits, we generated STZ-induced insulin-deficient T1DM mice. Two weeks after STZ injection, STZ mice exhibited weight loss, elevated blood glucose levels (>400 mg/dL), and low insulin levels . While tSubsequently, we examined the impact of STZ-induced T1DM on the phosphorylation of IRS1 at Ser residues and downstream components in the hippocampus. In STZ mice that had already developed memory impairment, the phosphorylation levels of hippocampal IRS1 at Ser residues in STZ mice were comparable to those in WT mice . AlthougAlthough memory decline associated with physiological aging can occur naturally, in contrast to pathogenic memory deficits, whether the onset processes of age-related memory impairment are related to the mechanisms underlying pathogenic memory deficits in T2DM and AD remains unclear. We examined the modification of IRS1 at Ser residues and the activity of downstream factors in the hippocampi of 21-month-old mice (aged mice). Aged mice displayed memory impairments without an increase in blood glucose and plasma insulin levels, despite an increase in body weight . MeanwhiIn common with DIO mice, the activation of mSer307, but not of mSer1097, on IRS1 accompanied by p70S6K activation and unchanged AMPK was observed with increased phosphorylation of IRS1 at mSer612 and mSer632/635 in the hippocampi of aged mice . NonetheNL-G-F mice carrying a humanized Aβ sequence and three AD mutations, i.e., Swedish, Beyreuther/Iberian, and Arctic mutations, in the endogenous App gene [NL-G-F mice. There were no differences in terms of body weight, blood glucose level, and plasma insulin concentration between WT and APPKINL-G-F mice at 12 weeks of age  and Saito et al. (2014). Similarly, human/rat/mouse Aβ40 and human Aβ40 sandwich ELISA revealed the same range for Aβ40 level in the T-PER fractions of the hippocampi of young APPKINL-G-F mice -based sandwich ELISA for human/rat/mouse Aβ42, we successfully confirmed that T-PER-extractable Aβ42 level had already increased in young APP KIG-F mice C, right.G-F mice D, right.NL-G-F mice. Importantly, the phosphorylation of hippocampal IRS1 at mSer307, mSer612, and mSer1097, but not at mSer632/635, significantly increased in 12-week-old APPKINL-G-F mice . However, the water T-maze test demonstrated that middle-aged APPKINL-G-F mice exhibited memory decline after 34 weeks under our experimental conditions (NL-G-F mice (right in NL-G-F mice, which were comparable to TBS-extractable Aβ42 levels in the brain and cortex of APPKINL-G-F mice [Although the onset of memory deficits was reported by 6 months in APPKIG-F mice , 6-monthnditions B. Meanwhright in C, D and G-F mice .NL-G-F mice showing memory decline (NL-G-F mice (NL-G-F mice (While an age-related increase in the phosphorylation of IRS1 at mSer612 and mSer632/635 accompanied by sustained phosphorylation of IRS1 at T2DM-related mSer1097 site and of AMPK was observed in the hippocampi of middle-aged APPKI decline , there wG-F mice E,F owingG-F mice D,E. RegaG-F mice . Our datThe present study demonstrates that memory decline in T1DM and T2DM in middle age occurs in an Aβ-independent manner, which is consistent with the findings of previous studies ,35,36. SNL-G-F mice, a concomitant increase in the basal phosphorylation of hippocampal IRS1 at multiple Ser sites with the activation of AMPK and elevation of Aβ42 level had already arisen at a young age, before the onset of memory decline, and that memory decline in middle age was accompanied by the persistence of these changes with age-related increase in the phosphorylation of IRS1 at specific Ser sites and in Aβ42 level.In middle-aged DIO and aged mice, Aβ-unrelated memory deficits are accompanied by increased basal phosphorylation of hippocampal IRS1 at specific Ser residues, whereas Aβ-unrelated memory impairments develop in STZ mice without the modification of IRS1. Although p70S6K activation accompanied by unchanged AMPK activity in the hippocampus is mutually observed with Aβ-unrelated memory deficits in middle-aged DIO, STZ, and aged mice regardless of the presence or absence of the phosphorylation of IRS1 at Ser residues, different alterations in other downstream components were observed in these mouse models. Furthermore, we found that, in APPKIHFD and genetically obese animals exhibit increased phosphorylation at mSer307 and p70S6K-induced phosphorylation at mSer1097 in peripheral tissues ,38. In tAdditionally, HFD- and STZ-induced diabetes increases the phosphorylation of JNK accompanied by the activation of mSer307 on IRS1 in the brain ,32. The NL-G-F mice showing normal metabolism and memory function displayed increased phosphorylation of hippocampal IRS1 at three Ser residues including mSer307, mSer612, and mSer1097, in which the Aβ42 level had already increased before the onset of memory decline. Consistent with previous studies [NL-G-F mice exhibiting memory decline with age-associated increase in the Aβ42 level.Although a link between the phosphorylation of neural IRS1 at mSer307, mSer612, and mSer632/635 and brain insulin resistance has been proposed ,16,43, b studies ,14,15,16NL-G-F mice, the concomitant phosphorylation of hippocampal IRS1 at mSer612 and mSer1097 with the activation of AMPK is constantly observed regardless of the presence or absence of the activation of mSer307 or mSer 632/635, suggesting that persistent phosphorylation of mSer612 and mSer1097 accompanied by AMPK activation may contribute to memory dysfunction in AD.Interestingly, in both young and middle-aged APPKINL-G-F mice reduces immunoreactivity in ELISA because the location of this mutation on the Aβ sequences is overlapped with the binding region of monoclonal antibody to Aβ used in human/rat/mouse Aβ sandwich ELISA [NL-G-F mice and that the Aβ42 level increased in middle-aged APPKINL-G-F mice. Owing to the elevation of Aβ42 level in the hippocampi of APPKINL-G-F mice from a young age, it is likely that the increased phosphorylation of IRS1 at Ser sites is associated with Aβ42 level but not with memory decline. Consistent with this finding, a previous study showed that the phosphorylation of IRS1 at mSer307/hSer312 and mSer612/hSer616 increased in cultured hippocampal neurons exposed to Aβ oligomers prepared from synthetic Aβ1–42 peptide and in the AβO-injected hippocampi of non-human primates [NL-G-F mice is accompanied by AMPK activation regardless of the presence or absence of memory decline. Consistent with our findings, recent studies suggest that AMPK activation is implicated in brain aging and development of neurodegenerative diseases, including AD [NL-G-F mice.Reportedly, the Arctic mutation in APPKIch ELISA ; howeveruding AD ,45. ThesIn summary, whether the modification of IRS1 through its phosphorylation at Ser sites in the hippocampus has a pathogenic or an adaptive function remains unknown because the phosphorylation of hippocampal IRS1 at mSer307, mSer612, and mSer1097 increased when metformin improves memory deficits in middle-aged DIO mice ,46. StudOur findings indicate that the phosphorylation of IRS1 at disease-specific Ser residues in the hippocampus may be a potential marker of Aβ-unrelated memory impairments induced by T2DM and aging. Alternatively, in Aβ-related memory decline in AD, the modification of IRS1 via its phosphorylation at multiple Ser sites accompanied by the activation of AMPK, a sensor of energy metabolism, may be a marker of the response to an early detection of elevated Aβ42 level before the onset of memory decline in AD.


LRRK2 G2019S PD patients and healthy individuals. Upon co-culture on top of PD astrocytes, control vmDAns displayed morphological signs of neurodegeneration and abnormal, astrocyte-derived α-synuclein accumulation. Conversely, control astrocytes partially prevented the appearance of disease-related phenotypes in PD vmDAns. We additionally identified dysfunctional chaperone-mediated autophagy (CMA), impaired macroautophagy, and progressive α-synuclein accumulation in PD astrocytes. Finally, chemical enhancement of CMA protected PD astrocytes and vmDAns via the clearance of α-synuclein accumulation. Our findings unveil a crucial non-cell-autonomous contribution of astrocytes during PD pathogenesis, and open the path to exploring novel therapeutic strategies aimed at blocking the pathogenic cross talk between neurons and glial cells.Parkinson's disease (PD) is associated with the degeneration of ventral midbrain dopaminergic neurons (vmDAns) and the accumulation of toxic α-synuclein. A non-cell-autonomous contribution, in particular of astrocytes, during PD pathogenesis has been suggested by observational studies, but remains to be experimentally tested. Here, we generated induced pluripotent stem cell-derived astrocytes and neurons from familial mutant   •Astrocytes contribute to dopaminergic neurodegeneration in PD•Non-cell-autonomous damage is triggered by impaired autophagy in PD astrocytes•Dysfunctional PD astrocytes accumulate and transfer α-synuclein to healthy DAns•CMA activator drug prevents α-synuclein accumulation and neurodegeneration In this article, Consiglio and colleagues show that PD iPSC-derived astrocytes contribute to dopaminergic neurodegeneration, indicating an important role for astrocytes in the pathogenesis of Parkinson's disease. PD astrocytes display dysfunctional chaperone-mediated autophagy (CMA), impaired macroautophagy, and progressive α-synuclein accumulation. In co-culture, PD astrocytes transfer α-synuclein to vmDAns and trigger dopaminergic neuronal cell death that can be rescued by treatment with a chemical enhancement of CMA. LRRK2), causing an autosomal dominant form of PD, account for 5% of familial cases and 2% of sporadic cases (Parkinson's disease (PD) is the second most prevalent neurodegenerative disease after Alzheimer's disease, affecting 7 to 10 million people worldwide . PD is cic cases . LRRK2 iic cases .Correlations between mutant LRRK2 and several pathogenic mechanisms linked to PD progression have been previously reported, including alterations in autophagy and consequent accumulation of α-syn . NeuronaLRRK2 mutations, and described the appearance of disease-specific phenotypes in iPSC-derived neurons, including impaired axonal outgrowth and deficient autophagic vacuole clearance  and two healthy age-matched controls (Ctrl SP09 and Ctrl SP17)  see A. No eviP17) see A and 1B.in iPSCs A–S1C.Tabwww.brainrnaseq.org/) in our iPSC-derived astrocytes, through RNA sequencing. We found that the transcriptomic profile of both Ctrl and PD iPSC-derived astrocytes was closer to that of human primary astrocytes than to that of their corresponding iPSC line, thus confirming their identity , whereas Ctrl vmDAns were obtained from two independent iPSC clones (Ctrl SP11 and Ctrl SP11#4) from a healthy age-matched control , and displayed spontaneous action potential firing  did not significantly change in numbers or morphology after co-culture with Ctrl or PD astrocytes  after 50 days of culture, which are not evident in Ctrl vmDAns . To testLRRK2 G2019S in Ctrl astrocytes. In these experiments, Ctrl astrocytes were transfected with a plasmid expressing V5-tagged LRRK2 G2019S, or with a Ctrl plasmid expressing GFP, and analyzed 7 days after transfection for the presence of α-syn. Astrocytes transfected with LRRK2 G2019S exhibited diffuse cytoplasmic accumulations of α-syn , and fully characterized these cells  targeting and silencing the LAMP2A spliced transcript (shLAMP2A), or an shRNA targeting the Luciferase gene (shLuc) as a control F. The shSince PD astrocytes displayed higher levels of α-syn compared with Ctrl astrocytes, we next investigated possible differences in α-syn turnover in these cells. α-syn has previously been shown to undergo degradation both by the ubiquitin/proteasome system and by autophagy ; therefoCells often respond to blockage in CMA by upregulating other autophagic pathways such as macroautophagy . However4Cl, inhibitors of lysosomal proteolysis, to inhibit LC3-II degradation. Under these conditions, PD astrocytes exhibited lower increase in LC3-II levels compared with controls, suggesting an impairment of the autophagy flux in PD astrocytes  and the autophagic cargo p62, and reduced autophagic flux (for both LC3-II and p62). The lower co-localization between the autophagosomal and lysosomal markers observed in PD astrocytes suggests that the reduced autophagic flux is due to a defect in autophagosome/lysosome fusion, similar to that previously described in PD neurons.Taking into account the coordinate functioning of the proteolytic systems, and the fact that CMA disruption seems to occur early during the development of PD pathology, we attempted to restore normal α-syn proteostasis by enhancing CMA activity. Our findings in cells treated with a chemical activator of CMA suggest that upregulation of CMA is still possible in these cells and that this intervention is sufficient to return levels of α-syn close to those in Ctrl cells. Although α-syn was cleared, restoration during a co-culture with Ctrl neurons was only partial in terms of neurite length and number, suggesting that the neurodegeneration observed could also be due to other non-α-syn-related factors secreted by PD astrocytes.Overall, our findings propose a specific role for astrocytes in mediating dopaminergic cell death during PD. PD-specific phenotypes specifically related to dysfunctions in the pathways of protein degradation have been observed in PD astrocytes and not in Ctrl astrocytes. Dysfunctional CMA, progressive α-syn accumulation, and glia-to-neuron transfer found in our PD astrocytes are all aspects that can compromise neuronal survival during PD pathogenesis. Future studies will identify whether additional factors other than α-syn are being secreted by (or lacking in) PD astrocytes, and thus contributing to triggering vmDA neuronal cell death. iPSC-based technology allows for the proper recapitulation of patient-specific disease-related phenotypes, which will aid in the discovery of new therapies.Experimental procedures are also provided in The parental iPSC lines used in our studies were previously generated and fully characterized . The genFour different iPSCs, two PD (SP12 and SP13) and two Ctrl (SP11 and SP11#4), were differentiated into dopaminergic neurons using a combination of two previously published protocols for midbrain induction . DetaileICC on cell cultures was performed as described in ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001), and the mean and standard error of the mean were plotted using Prism (Mac OS X). Number of independent experiments (n) is indicated in each figure legend.Statistical analyses of the obtained data were performed using two-tailed unequal variance Student’s t tests and ANOVA (Conceptualization, A.C. and A.R.; Methodology, I.F., J.P.M., A.Z., A.M.C., and J.S.; Investigation, Y.R.P, A.D., G.C., C.C., M.P.-E., M.G., I.F.-C., J.P., and A.F.; Validation, A.C. and A.R.; Writing – Original Draft, A.D.; Writing – Review & Editing, A.C. and A.R.; Visualization, A.D.; Resources, A.Z., A.M.C., J.S., E.T., A.C., and A.R.; Funding Acquisition, A.C.; Supervision, A.C.


The mammalian homologs of Lin-28, Lin28  and Lin28B, are promising cancer biomarkers. This meta-analysis was performed to evaluate the prognostic values of Lin28A and Lin28B in multiple human malignancies.Systematic searches of the PubMed, Web of Science and Embase were used to identify relevant studies. Pooled hazard ratios (HRs) with 95% confidence intervals (CI) for overall survival (OS), recurrence-free survival (RFS), disease-free survival (DFS), or progression-free survival (PFS) were respectively calculated.3772 Lin28A-associated patients and 1730 Lin28B-related cases were ultimately enrolled in this meta-analysis. The elevated expression level of Lin28A was significantly associated with poor OS  and poor RFS/DFS/PFS  in patients with malignancies. Lin28B overexpression significantly correlated with unfavorable OS  and RFS/DFS/PFS  of human malignancies.Lin28A and Lin28B possess significant prognostic values in various human malignancies. Overexpression of Lin28A or Lin28B suggests poor prognosis for cancer patients.The online version of this article (10.1186/s12935-019-0788-z) contains supplementary material, which is available to authorized users. In recent years, cancer has become the primary cause of mortality in most countries and regions, and the incidence of human malignancies has increased substantially . Given tLin28A and Lin28B are highly conserved RNA-binding and microRNA-regulated proteins. In general, they selectively block the expression of let-7 microRNA family members, which act as tumor suppressors by inhibiting the expression of oncogenes and key regulators of mitogenic pathways, including RAS, MYC, and HMGA2 . Many stOverexpression of Lin28A and Lin28B is associated with poor prognosis in various cancers, such as oral squamous cell carcinoma (OSCC) , 7, coloWe carefully performed a comprehensive search on PubMed, Web of Science, and Embase until June 2017 to identify relevant potential literatures. The following set of keywords was simultaneously applied for the online study search: “Lin28” or “Lin28” or “Lin28A” or “Lin28B” and “tumor” or “tumour” or “cancer” or “carcinoma” or “neoplasm” or “malignancies.”This meta-analysis was strictly conducted in accordance with the guidelines of the preferred reporting items of systematic reviews and meta-analyses (PRISMA) statement . StudiesThe extracted data included the following elements: (i) first author and publication year; (ii) characteristics of the studied population, including patients’ nationality, region, mean or median age, study size, disease type, and examined type of sample; (iii) cut-off definition and method of sample analysis; (iv) source of HR, mean, or median follow-up duration; and (v) HRs of elevated Lin28A or Lin28B expression for OS, recurrence-free survival (RFS), disease-free survival (DFS), or progression-free survival (PFS). If HRs were not directly reported, then the survival data were extracted from Kaplan–Meier plots by using Engauge Digitizer V.5.1 . Furthermore, HRs with 95% confidence intervals (CIs) were calculated using practical methods by Stata V.12.0  [P value) and Higgins I2 statistic. Sensitivity analyses, including influence analysis and Galbraith plot, were implemented for individual studies to identify the source of heterogeneity. If statistically significant heterogeneity was observed (P < 0.10 or I2 > 50%), the random-effects model was applied to estimate the pooled HR; otherwise, the fixed-effects model was used. If necessary, we also classified the included studies into subgroups on the basis of similar characteristics to minimize the influence of significant heterogeneity. In addition, publication bias was estimated using Egger’s test with funnel plot. Stata version 12.0  was used to calculate all statistical analyses. All P values were two sided, and a P value of less than 0.05 was considered statistically significant.In this meta-analysis, HRs and corresponding 95% CIs were combined to estimate the prognostic values of high Lin28A or Lin28B expression for cancer prognosis. An individual or pooled HR of more than 1.0 indicated poor prognosis for patients with Lin28A or Lin28B overexpression, and an HR of less than 1.0 represented better prognosis. Moreover, a fixed-effects model or a random-effects model was applied for meta-analysis on the basis of the heterogeneity among the pooled studies . HeterogAs shown in Fig. The main characteristics and data of the included studies are summarized in Tables I2= 93.7%, P < 0.001) . The pooled HR value of Lin28A overexpression for OS was 1.60  (P < 0.001), as detected by the fixed-effects model  . The fixed-effects model was applied, and the pooled HR value of Lin28A overexpression for RFS/DFS/PFS was 1.62  (P < 0.001)  , and the pooled HR detected by the fixed-effects model demonstrated that high Lin28B expression significantly correlated with poor OS of human malignancies  (P < 0.001)    and EOC   and North America   were first identified through mutagenesis screening as heterochronic genes in  elegans , 34, and elegans . Recent  elegans . Althoug elegans –40. As o elegans , 7, colo elegans , EOC [4, elegans , 11, gas elegans –14, HCC  elegans –17, and  elegans , 19. Alt elegans . Many of elegans , and IGF elegans . TherefoIn this meta-analysis, we investigated the prognostic values of Lin28A and Lin28B for multiple human malignancies. Combining the outcomes of studies regarding the association of lin28 expression and tumor prognosis, we have successfully drawn many valuable results. First, increased expression level of Lin28A was considered to predict poor OS and RFS/DFS/PFS for cancer patients, with combined HR values of 1.60  and 1.62 , respectively. Second, considering the pooled outcomes of studies on the relation between Lin28B expression and cancer prognosis, we found that elevated Lin28B level was significantly associated with poor OS and RFS/DFS/PFS of malignant diseases, with pooled HR values of 1.72  and 2.35 , correspondingly, which also exerted statistical significance. Our findings suggested that Lin28A and Lin28B are promising biomarkers, and the detection of Lin28A and Lin28B expression in cancer patients is of potential value for monitoring patients’ survival.I2 = 69.2%, P = 0.006). In subgroup analyses, no heterogeneity was observed in the subtotals of Chinese Taiwan  and EOC . Also, no heterogeneity was shown in the subgroup analyses by different assay methods for Lin28A overexpression and OS, and RFS/DFS/PFS   and North America  showed significant heterogeneity  and cancer prognosis.Additional file 2: Table S2. Main characteristics of studies on Lin28B and cancer prognosis.Additional file 3: Figure S1. In initial meta-analysis, forest plots summarizing the association of Lin28A overexpression and OS (A), and RFS/DFS/PFS (B) in patients with various cancers.Additional file 4: Figure S2. Influence analysis and Galbraith plot of individual studies on Lin28A expression and OS. A, B Influence analysis and Galbraith plot for initial meta-analysis; C, D influence analysis and Galbraith plot after study exclusion.Additional file 5: Figure S3. Influence analysis and Galbraith plot of individual studies on Lin28A expression and RFS/DFS/PFS. A, B Influence analysis and Galbraith plot for initial meta-analysis; C, D influence analysis and galbraith plot after study exclusion.Additional file 6: Figure S4. In initial meta-analysis, forest plots summarizing the association of Lin28B overexpression and OS (A), and RFS/DFS/PFS (B) in patients with various cancers.Additional file 7: Figure S5. Influence analysis and Galbraith plot of individual studies on Lin28B expression and OS. A, B Influence analysis and Galbraith plot for initial meta-analysis; C, D influence analysis and Galbraith plot after study exclusion.Additional file 8: Figure S6. Influence analysis and Galbraith plot of individual studies on Lin28B expression and RFS/DFS/PFS. A, B Influence analysis and Galbraith plot for initial meta-analysis; C, D influence analysis and Galbraith plot after study exclusion.Additional file 9: Figure S7. Forest plots evaluating the association of Lin28B overexpression and RFS/DFS/PFS in subgroups of Chinese Taiwan and EOC. HR, hazard ratio; CI, confidence interval; EOC, epithelial ovarian carcinoma.Additional file 10: Figure S8. Forest plots evaluating the association of Lin28B overexpression and RFS/DFS/PFS in subgroups of Asian and North America.Additional file 11: Figure S9. Subgroup analyses by different assay methods evaluating the association of (A) Lin28A overexpression and OS, (B) Lin28A overexpression and RFS/DFS/PFS, (C) Lin28B overexpression and OS, (D) Lin28B overexpression and RFS/DFS/PFS.