A novel pyruvate kinase and its application in lactic acid production under oxygen deprivation in Corynebacterium glutamicum

BACKGROUND: Pyruvate kinase (Pyk) catalyzes the generation of pyruvate and ATP in glycolysis and functions as a key switch in the regulation of carbon flux distribution. Both the substrates and products of Pyk are involved in the tricarboxylic acid cycle, anaplerosis and energy anabolism, which places Pyk at a primary metabolic intersection. Pyks are highly conserved in most bacteria and lower eukaryotes. Corynebacterium glutamicum is an industrial workhorse for the production of various amino acids and organic acids. Although C. glutamicum was assumed to possess only one Pyk (pyk1, NCgl2008), NCgl2809 was annotated as a pyruvate kinase with an unknown role. RESULTS: Here, we identified that NCgl2809 was a novel pyruvate kinase (pyk2) in C. glutamicum. Complementation of the WTΔpyk1Δpyk2 strain with the pyk2 gene restored its growth on d-ribose, which demonstrated that Pyk2 could substitute for Pyk1 in vivo. Pyk2 was co-dependent on Mn(2+) and K(+) and had a higher affinity for ADP than phosphoenolpyruvate (PEP). The catalytic activity of Pyk2 was allosterically regulated by fructose 1,6-bisphosphate (FBP) activation and ATP inhibition. Furthermore, pyk2 and ldhA, which encodes l-lactate dehydrogenase, were co-transcribed as a bicistronic mRNA under aerobic conditions and pyk2 deficiency had a slight effect on the intracellular activity of Pyk. However, the mRNA level of pyk2 in the wild-type strain under oxygen deprivation was 14.24-fold higher than that under aerobic conditions. Under oxygen deprivation, pyk1 or pyk2 deficiency decreased the generation of lactic acid, and the overexpression of either pyk1 or pyk2 increased the production of lactic acid as the activity of Pyk increased. Fed-batch fermentation of the pyk2-overexpressing WTΔpyk1 strain produced 60.27 ± 1.40 g/L of lactic acid, which was a 47% increase compared to the parent strain under oxygen deprivation. CONCLUSIONS: Pyk2 functioned as a pyruvate kinase and contributed to the increased level of Pyk activity under oxygen deprivation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12896-016-0313-6) contains supplementary material, which is available to authorized users

Integrated analysis of single-cell RNA-seq and chipset data unravels PANoptosis-related genes in sepsis

BackgroundThe poor prognosis of sepsis warrants the investigation of biomarkers for predicting the outcome. Several studies have indicated that PANoptosis exerts a critical role in tumor initiation and development. Nevertheless, the role of PANoptosis in sepsis has not been fully elucidated.MethodsWe obtained Sepsis samples and scRNA-seq data from the GEO database. PANoptosis-related genes were subjected to consensus clustering and functional enrichment analysis, followed by identification of differentially expressed genes and calculation of the PANoptosis score. A PANoptosis-based prognostic model was developed. In vitro experiments were performed to verify distinct PANoptosis-related genes. An external scRNA-seq dataset was used to verify cellular localization.ResultsUnsupervised clustering analysis using 16 PANoptosis-related genes identified three subtypes of sepsis. Kaplan-Meier analysis showed significant differences in patient survival among the subtypes, with different immune infiltration levels. Differential analysis of the subtypes identified 48 DEGs. Boruta algorithm PCA analysis identified 16 DEGs as PANoptosis-related signature genes. We developed PANscore based on these signature genes, which can distinguish different PANoptosis and clinical characteristics and may serve as a potential biomarker. Single-cell sequencing analysis identified six cell types, with high PANscore clustering relatively in B cells, and low PANscore in CD16+ and CD14+ monocytes and Megakaryocyte progenitors. ZBP1, XAF1, IFI44L, SOCS1, and PARP14 were relatively higher in cells with high PANscore.ConclusionWe developed a machine learning based Boruta algorithm for profiling PANoptosis related subgroups with in predicting survival and clinical features in the sepsis

Long-term survival, toxicities, and the role of chrono-chemotherapy with different infusion rates in locally advanced nasopharyngeal carcinoma patients treated with intensity-modulated radiation therapy: a retrospective study with a 5-year follow-up

PurposeThis study aimed to evaluate 5-year outcomes and the late toxicity profile of chrono-chemotherapy with different infusion rates in patients with locally advanced nasopharyngeal carcinoma (NPC).Methods and materialsOur retrospective analysis included 70 patients with locally advanced NPC stages III and IVB (according to the 2010 American Joint Committee on Cancer staging system). Patients were treated with two cycles of induction chemotherapy (IC) before concurrent chemoradiotherapy (CCRT) at Guizhou Cancer Hospital. The IC with docetaxel, cisplatin (DDP) and fluorouracil regimen. Patients were divided into two groups during CCRT. Using a “MELODIE” multi-channel programmed pump, DDP (100 mg/m2) was administered for 12 hours from 10:00 am to 10:00 pm and repeated every 3 weeks for 2-3 cycles. DDP was administered at the peak period of 4:00 pm in the sinusoidal chrono-modulated infusion group (Arm A, n=35). The patients in Arm B received a constant rate of infusion. Both arms received radiotherapy through the same technique and dose fraction. The long-term survival and disease progression were observed.ResultsAfter a median follow-up of 82.8 months, the 5-year progression-free survival rate was 81.3% in Arm A and 79.6% in Arm B (P = 0.85). The 5-year overall survival rate was not significantly different between Arm A and Arm B (79.6% vs 85.3%, P = 0.79). The 5-year distant metastasis-free survival rate was 83.6% in Arm A and 84.6% in Arm B (P = 0.75). The 5-year local recurrence-free survival rate was 88.2% in Arm A and 85.3% in Arm B (P = 0.16). There were no late toxicities of grade 3-4 in either group. Both groups had grade 1-2 late toxicities. Dry mouth was the most common late toxic side effect, followed by hearing loss and difficulty in swallowing. There was no statistically significant difference between Arm A and Arm B in terms of side effects.ConclusionLong-term analysis confirmed that in CCRT, cisplatin administration with sinusoidal chrono-modulated infusion was not superior to the constant infusion rate in terms of long-term toxicity and prognosis

MASALAH-MASALAH PEMBELAJARAN YANG DIHADAPI WIDYAISWARA : Studi Kasus Pada Lembaga Diktat Pemda Tk.I Propinsi Bengkulu

<div><p>Rat strains differ dramatically in their susceptibility to mammary carcinogenesis. On the assumption that susceptibility genes are conserved across mammalian species and hence inform human carcinogenesis, numerous investigators have used genetic linkage studies in rats to identify genes responsible for differential susceptibility to carcinogenesis. Using a genetic backcross between the resistant Copenhagen (Cop) and susceptible Fischer 344 (F344) strains, we mapped a novel mammary carcinoma susceptibility (<i>Mcs30</i>) locus to the centromeric region on chromosome 12 (LOD score of ∼8.6 at the D12Rat59 marker). The <i>Mcs30</i> locus comprises approximately 12 Mbp on the long arm of rat RNO12 whose synteny is conserved on human chromosome 13q12 to 13q13. After analyzing numerous genes comprising this locus, we identified <i>Fry</i>, the rat ortholog of the furry gene of <i>Drosophila melanogaster,</i> as a candidate <i>Mcs</i> gene. We cloned and determined the complete nucleotide sequence of the 13 kbp <i>Fry</i> mRNA. Sequence analysis indicated that the <i>Fry</i> gene was highly conserved across evolution, with 90% similarity of the predicted amino acid sequence among eutherian mammals. Comparison of the <i>Fry</i> sequence in the Cop and F344 strains identified two non-synonymous single nucleotide polymorphisms (SNPs), one of which creates a putative, de novo phosphorylation site. Further analysis showed that the expression of the <i>Fry</i> gene is reduced in a majority of rat mammary tumors. Our results also suggested that FRY activity was reduced in human breast carcinoma cell lines as a result of reduced levels or mutation. This study is the first to identify the <i>Fry</i> gene as a candidate <i>Mcs</i> gene. Our data suggest that the SNPs within the <i>Fry</i> gene contribute to the genetic susceptibility of the F344 rat strain to mammary carcinogenesis. These results provide the foundation for analyzing the role of the human <i>FRY</i> gene in cancer susceptibility and progression.</p></div

How Potential Evapotranspiration Regulates the Response of Canopy Transpiration to Soil Moisture and Leaf Area Index of the Boreal Larch Forest in China

Transpiration is a critical component of the hydrological cycle in the terrestrial forest ecosystem. However, how potential evapotranspiration regulates the response of canopy transpiration to soil moisture and leaf area index of the boreal larch forest in China has rarely been evaluated. The present study was conducted in the larch (Larix gmelinii (Rupr.) Rupr.) forest, which is a typical boreal forest in China. The canopy transpiration was measured using sap flow techniques from May to September in 2021 and simultaneously observing the meteorological variables, leaf area index (LAI) and soil moisture (SWC). The results showed that there were significant differences in canopy transpiration of Larix gmelinii among the months. The correlation and regression analysis indicated that canopy transpiration was mainly influenced by potential evapotranspiration (PET), while the effect of soil moisture on canopy transpiration was lowest compared with other environmental factors. Furthermore, our results revealed that the effect of PET on canopy transpiration was not regulated by soil moisture when soil moisture exceeded 0.2 cm3 cm−3. More importantly, under the condition of sufficient soil moisture, it was demonstrated that the response of canopy transpiration to leaf area index was limited when PET exceeded 9 mm/day. These results provide valuable implications for supporting forest management and water resource utilization in the boreal forest ecosystem under the context of global warming.</jats:p