PI3Kγ maintains the self-renewal of acute myeloid leukemia stem cells by regulating the pentose phosphate pathway.

ABSTRACT: Acute myeloid leukemia (AML) is an aggressive hematological malignancy originating from transformed hematopoietic stem or progenitor cells. AML prognosis remains poor owing to resistance and relapse driven by leukemia stem cells (LSCs). Targeting molecules essential for LSC function is a promising therapeutic approach. The phosphatidylinositol 3-kinase (PI3K)/AKT pathway is often dysregulated in AML. We found that although PI3Kγ is highly enriched in LSCs and critical for self-renewal, it was dispensable for normal hematopoietic stem cells. Mechanistically, PI3Kγ-AKT signaling promotes nuclear factor erythroid 2-related factor 2 (NRF2) nuclear accumulation, which induces 6-phosphogluconate dehydrogenase (PGD) and the pentose phosphate pathway, thereby maintaining LSC stemness. Importantly, genetic or pharmacological inhibition of PI3Kγ impaired expansion and stemness of murine and human AML cells in vitro and in vivo. Together, our findings reveal a key role for PI3Kγ in selectively maintaining LSC function by regulating AKT-NRF2-PGD metabolic pathway. Targeting the PI3Kγ pathway may, therefore, eliminate LSCs without damaging normal hematopoiesis, providing a promising therapeutic strategy for AML.

STING agonism overcomes STAT3-mediated immunosuppression and adaptive resistance to PARP inhibition in ovarian cancer.

BACKGROUND: Poly (ADP-ribose) polymerase (PARP) inhibition (PARPi) has demonstrated potent therapeutic efficacy in patients with BRCA-mutant ovarian cancer. However, acquired resistance to PARPi remains a major challenge in the clinic. METHODS: PARPi-resistant ovarian cancer mouse models were generated by long-term treatment of olaparib in syngeneic Brca1-deficient ovarian tumors. Signal transducer and activator of transcription 3 (STAT3)-mediated immunosuppression was investigated in vitro by co-culture experiments and in vivo by analysis of immune cells in the tumor microenvironment (TME) of human and mouse PARPi-resistant tumors. Whole genome transcriptome analysis was performed to assess the antitumor immunomodulatory effect of STING (stimulator of interferon genes) agonists on myeloid cells in the TME of PARPi-resistant ovarian tumors. A STING agonist was used to overcome STAT3-mediated immunosuppression and acquired PARPi resistance in syngeneic and patient-derived xenografts models of ovarian cancer. RESULTS: In this study, we uncover an adaptive resistance mechanism to PARP inhibition mediated by tumor-associated macrophages (TAMs) in the TME. Markedly increased populations of protumor macrophages are found in BRCA-deficient ovarian tumors that rendered resistance to PARPi in both murine models and patients. Mechanistically, PARP inhibition elevates the STAT3 signaling pathway in tumor cells, which in turn promotes protumor polarization of TAMs. STAT3 ablation in tumor cells mitigates polarization of protumor macrophages and increases tumor-infiltrating T cells on PARP inhibition. These findings are corroborated in patient-derived, PARPi-resistant BRCA1-mutant ovarian tumors. Importantly, STING agonists reshape the immunosuppressive TME by reprogramming myeloid cells and overcome the TME-dependent adaptive resistance to PARPi in ovarian cancer. This effect is further enhanced by addition of the programmed cell death protein-1 blockade. CONCLUSIONS: We elucidate an adaptive immunosuppression mechanism rendering resistance to PARPi in BRCA1-mutant ovarian tumors. This is mediated by enrichment of protumor TAMs propelled by PARPi-induced STAT3 activation in tumor cells. We also provide a new strategy to reshape the immunosuppressive TME with STING agonists and overcome PARPi resistance in ovarian cancer.

Profile of Folate in Breast Milk from Chinese Women over 1-400 Days Postpartum.

Folate is an essential nutrient for growth in early life. This study aimed to determine the levels and compositions of folate in Chinese breast milk samples. This study was part of the Maternal Nutrition and Infant Investigation (MUAI) study. A total of 205 healthy mothers were randomly recruited in Chengdu over 1−400 days postpartum. Five different species of folate, including tetrahydrofolate (THF), 5-methyl-THF, 5,10-methenyl-THF,5-formyl-THF and unmetabolized folic acid (UMFA), were measured for liquid chromatography−tandem mass spectrometry (LC-MS). The median levels of total folate ranged from 12.86 to 56.77 ng/mL in the breast milk of mothers at 1−400 days postpartum, gradually increasing throughout the lactating periods. The median levels of 5-methyl-THF, minor reduced folate (the sum of THF, 5,10-methenyl-THF and 5-formyl-THF) and UMFA were in the ranges of 8.52−40.65 ng/mL, 3.48−16.15 ng/mL and 0.00−1.24 ng/mL during 1−400 days postpartum, respectively. 5-Methyl-THF accounted for more than 65% of the total folate in all breast milk samples. The levels of UMFA in mature breast milk samples were higher in supplement users than nonusers, but not for colostrum and transitional milk samples (p < 0.05). In conclusion, the level of total folate in the breast milk changed along with the prolonged lactating periods, but 5-methyl-THF remains the dominant species of folate in the breast milk of Chinese populations across all entire lactating periods.

[Fatty acid composition of human breast milk across lactational stages in six representative cities in China from 2018 to 2019].

OBJECTIVE: To investigate the fatty acid composition in breast milk at different lactation stages in six representative cities of China. METHODS: From January 2018 to December 2019, milk sampling of 690 healthy lactating mothers(full-term) in 5 lactation periods of 0-5 days, 10-14 days, 40-45 days, 200-240 days and 300-400 days was collected from 6 representative regions in China, with 23 cases of breast milk received in each lactation stage in each city. Mix it into one mixture, and make a total of 30 mixes. Determination of fatty acids in breast milk was conducted by gas chromatography-flame ionization detector. RESULTS: The contents of total fatty acids(TFA), saturated fatty acids(SFA), monounsaturated fatty acids(MUFA) and polyunsaturated fatty acids(PUFA) in breast milk increased with the progress of lactation and reached a relatively stable level after reaching a peak at 40-45 days. However, the composition ratio of SFA, MUFA and PUFA in TFA remained relatively stable from 0 to 400 days. The ratio of arachidonic to docosahexaenoic(AA/DHA) in breast milk from 0 to 400 days in the six cities ranged from 1.14 to 1.55, and there was no obvious trend of change in the whole lactation stage. The ratio of linoleic acid to alpha-linolenic acid(LA/ALA) in Chinese breast milk ranged from 3.84 to 18.94, showing significant regional variation. CONCLUSION: The content and composition of fatty acids in breast milk of six cities in China vary to a certain extent and show a dynamic change process with the passage of time of lactation.

[Feeding status of 0～23-month-old infants in poor rural areas of Gansu Province from 2018 to 2019].

OBJECTIVE: To investigate the feeding status of infants and their feeder's feeding literacy in poor rural areas of Gansu Province. METHODS: From November 2018 to January 2019, a multi-stage cluster random sampling method was used to select 1200 infant and child families aged 0 to 23 months in 40 villages of Gansu Province. A standardized questionnaire from the Chinese Nutrition Society(CNS)was used to investigate the basic situation of infant and young children's families, the situation of breastfeeding and the addition of supplementary food, and parents feeding knowledge, attitude behavior(KAP). Using chi-square test, logistic regression and other method to statistically describe and infer the collected data. RESULTS: A total of 1193 infants and 1165 feeders were investigated. The exclusive breastfeeding rate of infants and young children under the age of 6 months was 39. 02%. The rate of continuous breastfeeding at 1 year old was 37. 40%, and the rate of continuous breastfeeding at 2 years old was 20. 88%, the difference between the two was significant(χ~2=13. 498, P<0. 01). The supplementary food supplement rate of infants and children over 6 months was 94. 37%, the highest supplementary supplement for cereals and potatoes(98. 01%), and the lowest percentage for beans and nuts(23. 51%), and the distribution of supplementary foods at different ages was significantly different(χ~2=52. 336, P<0. 01). The qualification rate of infants and young children's minimum dietary diversity was 64. 13%, the minimum eating frequency qualification rate was 70. 64%, the minimum acceptable dietary intake qualification rate was 42. 16%, and the qualification rates of various indicators were significantly different between different months(χ~2=85. 421, P<0. 01;χ~2=19. 66, P<0. 01; χ~2=17. 261, P<0. 01). The KAP score passing rate of infant caregivers was 37. 34%, and there was a statistical difference between the age of infants and young children, the education level and the sex of the caregiver(χ~2=9. 411, P<0. 05;χ~2=25. 901, P<0. 01;χ~2=3. 874, P<0. 05). Taking low-month-old infants and young children, low education and male caregivers as controls, infants and young children aged over 12 months, high school education and female caregivers were the protective factors of KAP scores(P<0. 05). CONCLUSION: The problems of infant breastfeeding and supplementary feeding in poor rural areas of Gansu Province were serious, and the knowledge and skills of raising people were scarce, which were related to the age of infants and young children, the education and the sex of raising people.

Reporting, presentation and wording of recommendations in clinical practice guideline for gout: a systematic analysis.

OBJECTIVES: We systematically analysed recommendations from gout guidelines as an example, to provide a basis for developing a reporting standard of recommendations in clinical practice guidelines (CPGs). DESIGN: Systematic review without meta-analysis. METHODS: We systematically searched MEDLINE and all relevant guideline websites (National Institute for Health and Care Excellence, National Guideline Clearinghouse, Scottish Intercollegiate Guidelines Network, WHO, Guidelines International Network, DynaMed, UpTodate, Best Practice) from their inception to January 2017 to identify and select gout CPGs. We used search terms such as 'gout', 'hyperuricemia' and 'guideline'. We included the eligible CPGs of gout according to the predefined inclusion and exclusion criteria after screening titles, abstracts and full texts. The characteristics of recommendations reported in the included guidelines were extracted and analysed. RESULTS: A total of 15 gout guidelines with a range of 5-80 recommendations were retrieved. Several indicators were used in the gout guidelines to facilitate identification of recommendations, including grouping all recommendations in a summary section, formatting recommendations in a particular or special way, using locating words for recommendations and indicating the strength of recommendation and quality of evidence. We found some components commonly used in the recommendations. The wording of recommendations varied across guidelines. Recommendations were detailed and explained in the section of rationale and explanation of recommendations. In some guidelines, recommendations were accompanied with other material to assist their reporting. CONCLUSIONS: Variability and inconsistency were found on the reporting and presentation of recommendations in gout guidelines. Several points for reporting recommendation can be summarised. First, we suggested summarising and highlighting the core recommendations in a guideline. Second, guideline developers should try to structure and write recommendations reasonably. Third, it was necessary to detail and explain the recommendations and their rationale. Finally, describing and providing other potential useful contents was also a helpful way for clear reporting.

Targeted Profiling of RNA Translation.

This unit describes a reverse transcription-quantitative PCR (RT-qPCR)-based method for gene-targeted measurement of RNA translation levels. The method includes washing and lysing cells with a buffer containing cycloheximide to enrich ribosomal accumulation at translation initiation sites (TIS), followed by enzymatic treatment to generate ribosomal footprints, reverse transcription targeted towards TIS of specific transcripts of interest to generate complementary DNA (cDNA), and qPCR to measure the abundance of these footprints. This method enables time- and cost-effective assessment of changes in translation levels across focused panels of genes and across numerous samples. © 2018 by John Wiley & Sons, Inc.

Targeted profiling of RNA translation reveals mTOR-4EBP1/2-independent translation regulation of mRNAs encoding ribosomal proteins.

The PI3K-Akt-mTOR signaling pathway is a master regulator of RNA translation. Pharmacological inhibition of this pathway preferentially and coordinately suppresses, in a 4EBP1/2-dependent manner, translation of mRNAs encoding ribosomal proteins. However, it is unclear whether mechanistic target of rapamycin (mTOR)-4EBP1/2 is the exclusive translation regulator of this group of genes, and furthermore, systematic searches for novel translation modulators have been immensely challenging because of difficulties in scaling existing RNA translation profiling assays. Here, we developed a rapid and highly scalable approach for gene-specific quantitation of RNA translation, termed Targeted Profiling of RNA Translation (TPRT). We applied this technique in a chemical screen for translation modulators, and identified numerous preclinical and clinical therapeutic compounds, with diverse nominal targets, that preferentially suppress translation of ribosomal proteins. Surprisingly, some of these compounds act in a manner that bypasses canonical regulation by mTOR-4EBP1/2. Instead, these compounds exert their translation effects in a manner that is dependent on GCN2-eIF2α, a central signaling axis within the integrated stress response. Furthermore, we were also able to identify metabolic perturbations that also suppress ribosomal protein translation in an mTOR-independent manner. Together, we describe a translation assay that is directly applicable to large-scale RNA translation studies, and that enabled us to identify a noncanonical, mTOR-independent mode for translation regulation of ribosomal proteins.

Engineering a high-performance, metagenomic-derived novel xylanase with improved soluble protein yield and thermostability.

The novel termite gut metagenomic-derived GH11 xylanase gene xyl7 was expressed in Escherichia coli BL21, and the purified XYL7 enzyme exhibited high specific activity (6340U/mg) and broad pH active range of 5.5-10.0. Directed evolution was employed to enhance the thermostability of XYL7; two mutants (XYL7-TC and XYL7-TS) showed a 250-fold increase in half-life at 55°C, with a 10°C increase in optimal temperature compared to that of wild-type XYL7. A truncated enzyme (XYL7-Tr3) acquired by protein engineering showed similar catalytic properties as the wild-type, with a tenfold increase in soluble protein yield by the mutant. The reducing sugar produced by XYL7-TC was about fourfold greater than that produced by their parents when incubated with xylan at 60°C for 4h. The engineered novel xylanase exhibited superior enzymatic performance and showed promise as an excellent candidate for industrial application due to its high specific activity, stability and soluble protein yield.

Phylogenetic and functional analysis of gut microbiota of a fungus-growing higher termite: Bacteroidetes from higher termites are a rich source of ß-glucosidase genes.

Fungus-growing termites, their symbiotic fungi, and microbiota inhibiting their intestinal tract comprise a highly efficient cellulose-hydrolyzing system; however, little is known about the role of gut microbiota in this system. Twelve fosmid clones with ß-glucosidase activity were previously obtained by functionally screening a metagenomic library of a fungus-growing termite, Macrotermes annandalei. Ten contigs containing putative ß-glucosidase genes (bgl1-10) were assembled by sequencing data of these fosmid clones. All these contigs were binned to Bacteroidetes, and all these ß-glucosidase genes were phylogenetically closed to those from Bacteroides or Dysgonomonas. Six out of 10 ß-glucosidase genes had predicted signal peptides, indicating a transmembrane capability of these enzymes to mediate cellulose hydrolysis within the gut of the termites. To confirm the activities of these ß-glucosidase genes, three genes (bgl5, bgl7, and bgl9) were successfully expressed and purified. The optimal temperature and pH of these enzymes largely resembled the environment of the host's gut. The gut microbiota composition of the fungus-growing termite was also determined by 454 pyrosequencing, showing that Bacteroidetes was the most dominant phylum. The diversity and the enzyme properties of ß-glucosidases revealed in this study suggested that Bacteroidetes as the major member in fungus-growing termites contributed to cello-oligomer degradation in cellulose-hydrolyzing process and represented a rich source for ß-glucosidase genes.

Biochemical characterization and crystal structure of a GH10 xylanase from termite gut bacteria reveal a novel structural feature and significance of its bacterial Ig-like domain.

Bacterial Ig-like (Big) domains are commonly distributed in glycoside hydrolases (GH), but their structure and function remains undefined. Xylanase is a GH, and catalyzes the hydrolysis of the internal ß-xylosidic linkages of xylan. In this study, we report the molecular cloning, biochemical and biophysical characterization, and crystal structure of a termite gut bacterial xylanase, Xyl-ORF19, which was derived from gut bacteria of a wood-feeding termite (Globitermes brachycerastes). The protein architecture of Xyl-ORF19 reveals that it has two domains, a C-terminal GH10 catalytic domain and an N-terminal Big_2 non-catalytic domain. The catalytic domain folds in an (α/ß)8 barrel as most GH10 xylanases do, but it has two extra ß-strands. The non-catalytic domain is structurally similar to an immunoglobulin-like domain of intimins. The recombinant enzyme without the non-catalytic domain has fairly low catalytic activity, and is different from the full-length enzyme in kinetic parameters, pH and temperature profiles, which suggests the non-catalytic domain could affect the enzyme biochemical and biophysical properties as well as the role for enzyme localization. This study provides a molecular basis for future efforts in xylanase bioengineering.

Discovery of (hemi-) cellulase genes in a metagenomic library from a biogas digester using 454 pyrosequencing.

In this study, 341, 246, and 386 positive clones with endo-ß-1,4-glucanase, ß-glucosidase, and endo-ß-1,4-xylanase activities, respectively, were identified by screening from a metagenomic fosmid library constructed from a biogas digester. Subsequently, pools of 4, 10, and 16 positive clones were subjected to 454 pyrosequencing in different subruns. In total, 21 unique glycosyl hydrolase (GH) genes were predicted by bioinformatic analysis, which showed similarities to their nearest neighbors from 39 % to 72 %. In addition to bioinformatics prediction, nine GH genes were expressed and purified to identify their activity with four kinds of substrates. The activities of the most expressed proteins were consistent with their annotation based on bioinformatics prediction; however, three GH genes belonging to the GH5 family showed different activities from their annotation. An efficient acidic cellulase En1 had an optimal condition at 55 °C, pH 5.5, with a specific activity toward carboxymethylcellulose at 118 U/mg and K m at 12.8 g/L. This study demonstrated that there are diverse GHs in the biogas digester system with potential industrial application in lignocellulose hydrolysis, and their activities should be investigated with different substrates before their application. Additionally, pool sequencing of positive fosmid clones might be a cost-effective approach to obtain functional genes from metagenomic libraries.

Characterization of a novel thermostable ß-glucosidase from a metagenomic library of termite gut.

A novel ß-glucosidase-encoding gene, bgl-gs1, which was identified from a positive fosmid clone in a metagenomic library of the gut of Globitermes brachycerastes, [corrected] encodes a 455 amino acid polypeptide that contains a catalytic domain belonging to glycoside hydrolase family 1 (GH1). It was expressed in Escherichia coli BL21 (DE3) and the expression product showed a molecular mass of ∼51.7 kDa by SDS-PAGE. The optimal temperature and pH for the activity of the purified recombinant enzyme Bgl-gs1 with p-nitrophenyl-ß-D-glucoside (pNPG) were 90°C and 6.0, respectively. The specific activities of Bgl-gs1 on pNPG and salicin were 110 and 14U/mg of protein, respectively, and its K(m) values were 0.18 and 2.59 mM, respectively. The residual activity of Bgl-gs1 was maintained above 70% after the recombinant enzyme was incubated at 75°C and pH 6.0 for 2h, and its half-life at 90°C was approximately 1h in the presence of 4mM pNPG. Bgl-gs1 showed synergistic effect with either a crude enzyme mixture of the fungal strain Trichoderma reesei Rut-C30 or a fusion protein (TcE1) created from the cellobiohydrolase cbh1 gene of T. reesei and endoglucanase from Acidothermus cellulolyticus; 87 and 137% increases in hydrolytic efficiency were noted on microcrystalline cellulose, respectively. These results suggest that the thermostable ß-glucosidase Bgl-gs1 is a likely candidate for industrial applications.

Effect of key factors on hydrogen production from cellulose in a co-culture of Clostridium thermocellum and Clostridium thermopalmarium.

A cellulolytic, hydrogen-producing bacterium (Clostridiumthermocellum DSM 1237) and a non-cellulolytic, hydrogen-producing bacterium (Clostridiumthermopalmarium DSM 5974) were co-cultured at 55 degrees C, using cellulose as the sole substrate. At a low load of cellulose (filter paper, 4.5g/L), yeast extract had a significant effect on cellulose degradation and hydrogen production. The extent of cellulose utilization and hydrogen production displayed a linear relationship with the logarithm of the yeast extract concentration, and the optimal weight ratio of yeast extract to cellulose was 1:1. At a high load of filter paper (9g/L), an alkali chemical was required to maintain efficient cellulose degradation. As the KHCO3 concentration increased from 0 to 60mM, the utilized cellulose increased from 1.23g/L (13.5%) to 8.59g/L (94.3%), and maximum hydrogen production (1387ml/L of culture) occurred at 40mM KHCO(3). Increasing the inoculation ratio of C. thermopalmarium to C. thermocellum from 0.05:1 to 0.17:1 had little influence on hydrogen production, probably because of the limited availability of soluble sugar in the medium during the early stages of the co-culture.