Identification and validation of a novel cuproptosis signature for stratifying different prognostic, immune, metabolic, and therapeutic landscapes in pancreatic adenocarcinoma.

OBJECTIVE: Pancreatic adenocarcinoma (PAAD) is a highly malignant cancer that urgently needs more effective therapeutic strategies. The discovery of cuproptosis brings great inspiration for the treatment and clinical assessment of cancers. MATERIALS AND METHODS: A novel cuproptosis-related (CR) risk signature was constructed using the Lasso regression analysis. Its prognostic value was assessed via a series of survival analyses and validated in four GEO cohorts. The effects of CR risk signature on tumor immune microenvironment (TIM) were explored through CIBERSORT, ESTIMATE, and ssGSEA algorithms. Using GESA, we investigated its associations with various patterns of programmed cell death (PCD) and the metabolism process. The somatic mutation features of each CR-risk group were also probed using 'maftools' R package and cBioPortal database. The potential linkages between CR risk score and the efficacy of multiple therapeutic approaches were elucidated using tumor mutation burden, the expressions of immune checkpoints, the TIDE score, and the GDSC database. Finally, we ascertained the biofunctions of LIPT1 (Lipoyltransferase 1) in pancreatic cancer (PC) cells through immunohistochemistry, qPCR (quantitative polymerase chain reaction), colony formation, and Transwell assays. RESULTS: LIPT1, LIAS (lipoyl synthase), PDP1 (Pyruvate dehydrogenase phosphatase1), and GCSH (Glycine cleavage system H protein) constituted the CR risk signature. The CR risk signature possessed a high prognostic value and could improve the traditional prognostic model. Moreover, the CR risk score was indicative of the changes in infiltration levels of CD8+T cells and macrophages, whereas it was not associated with the enrichment of various PCD patterns and multiple metabolic processes. As for therapeutic correlation, CR risk score was a potential biomarker for predicting the efficacy of ICBs but failed in targeted drugs and chemotherapeutic agents. Through qPCR and immunohistochemistry detection in clinical samples, we confirmed that LIPT1 was significantly downregulated in pancreatic adenocarcinoma (PAAD) samples. Experiments in vitro revealed that silencing LIPT1 promoted the proliferation, migration, and invasion of PANC-1 and SW1990 cells. CONCLUSIONS: The novel CR risk signature contributed to the risk stratification of PAAD patients. Cuproptosis regulatory genes, well represented by LIPT1, provided new insights into PAAD treatment and assessment.

A Study on Neuroprotective Effects of Curcumin on the Diabetic Rat Brain.

The present study was aimed to study the neuroprotective therapeutic effect of curcumin on the male albino rat brain. Subarachnoid hemorrhage leads to severe mortality rate and morbidity, and oxidative stress is a crucial factor in subarachnoid hemorrhage. Therefore, we investigated the effect of curcumin on oxidative stress and glutamate and glutamate transporter-1 on a subarachnoid hemorrhage-induced male albino rats. The curcumin commonly used for the treatment and saline used for the control. Curcumin (10 mg/kg bwt) dissolved in saline and administered orally to the rats for one week. Glutamate, glutamate transporter-1, malondialdehyde (MDA), superoxide dismutase (SOD), catalase, glutathione reductase and lactate dehydrogenase (LDH) activities were determined. Glutamate level was lower in the curcumin-treated rats compared to their respective controls. Glutamate transporter-1 did not alter in the curcumin-treated rats compared to their controls. Glutamate transporter-1 protein expression is significantly reduced in the curcumin-treated rats. MDA levels decreased 18 and 29 % in the hippocampus and the cortex region respectively. SOD (17% and 32%), and catalase (19% and 24%) activities were increased in the curcumin-treated hippocampus and the cortex region respectively. Glutathione reductase (13% and 19%) and LDH (21% and 30%) activities were increased in the treated hippocampus and the cortex region respectively. The mRNA expression of NK-kB and TLR4 was significantly reduced following curcumin treatment. Taking all these data together, the curcumin found to be effective against oxidative stress and glutamate neurotoxicity in the male albino rats.

First Report of Pear Blossom Blast Caused by Pseudomonas syringae pv. syringae in China.

In the spring of 2006, a new bacterial disease was noted in pear orchards near Hangzhou, Zhejiang Province, China. The disease caused severe blossom blast on pears (Pyrus pyrifolia; cv. Cuiguan). Early symptoms of the disease included blackening of the calyx end of developing fruit, blackening of blossom clusters while leaves of affected blossom clusters appeared normal, or death of clusters consisting of both blossoms and leaves. Later, tips of twigs turned dark brown and died. No bacterial ooze was observed. Twelve bacterial isolates were recovered from ten samples of buds and blossoms. Six isolates were selected for identification. They were similar to those of the reference strains of Pseudomonas syringae pv. syringae LMG5570 and LMG 2230 from Belgium in phenotypic tests on the basis of the Biolog Microbial Identification System (version 4.2; Biolog Inc., Hayward, CA), pathogenicity tests, gas chromatographic analysis of fatty acid methyl esters (FAMEs) using the Microbial Identification System (MIDI Inc., Newark, DE) with aerobic bacterial library (TABA50), and electron microscopy (TEM, KYKY-1000B, Japan). All isolates tested were gram-negative, aerobic rods measuring 1.5 to 2.4 × 0.5 to 0.6 µm with 2 to 4 polar flagella. Fluorescent green diffusible pigment was produced on King's Medium B. Colonies were gray-white and slightly raised with smooth margins on nutrient agar. They produced levan on sucrose nutrient agar. A hypersensitive reaction was observed on tobacco cv. Benshi 24 h after inoculation. All isolates were identified as P. syringae pv. syringae with Biolog similarity index of 0.57 to 0.86 and FAME similarity index of 0.58 to 0.81. Identification as P. syringae pv. syringae was confirmed using 16S rDNA universal primers (2,3): 5'-AGA GTT TGA TCA TGG CTC AG-3' forward primer, 5'-ACG GTT ACC TTG TTA CGA CTT-3' reverse primer. The PCR fragments of the three isolates were sequenced and compared with sequences in GenBank. They had 99% similiarity with P. syringae pv. syringae 16S rRNA gene strain NCPPB 3869. Koch's postulates were conducted on buds of the original pear cultivar growing in pots and detached pear blossoms in flasks by spray inoculation with cell suspensions containing 108 CFU/ml of the six isolates at 18 to 22°C with two replications. The bacteria induced symptoms on buds and blossoms similar to those observed in the field. The bacterium was reisolated from symptomatic pear buds and internal ovary tissues. P. syringae pv. syringae was first reported in England as the cause of pear blossom blast in 1914 (1). After searching all the Chinese agricultural databases and major journals (National Knowledge Infrastructure database, Vip Chinese periodical database, Chinese wanfang database, China InfoBank, Scientia Agricultura Sinica, Acta Phytopathologica Sinica, Acta Phytophylacica Sinica, and Journal of Fruit Science), to our knowledge, this is the first report of pear blossom blast caused by P. syringae pv. syringae in China. The disease cycle on pear trees and the control strategies in the regions are being further studied. References: (1) B. P. Barker et al. Ann. Appl. Biol. 1:85, 1914. (2) U. Edward et al. Nucleic Acids Res. 17:7843,1989. (3) B. Li et al. J. Phytopathol. 34:141, 2006.

Serum-free culture conditions for cells capable of producing long-term survival in lethally irradiated mice.

The goal of ex vivo culture is to expand and/or differentiate cells in culture such that they retain their functional characteristics when reinfused into a patient. The studies presented here analyzed the use of culture conditions devoid of serum to expand murine hematopoietic stem cells. Bone marrow cells from male B6D2F1/J mice were cultured for up to 28 days in serum-free medium in the absence or presence of stem cell factor (SCF), GM-CSF or a combination of the two factors. Cells cultured for up to 21 days were assessed for granulocyte-macrophage colony-forming cells (GM-CFC), spleen colony-forming units, and cells responsible for short-term and long-term hematopoietic repopulation in lethally irradiated mice. Compared to initial seeding levels, the presence of SCF and GM-CSF increased total cell numbers 90-fold and GM-CFC numbers 42-fold over a 21-28 day culture period. Although spleen colony-forming unit cells did not increase, they were maintained at initial seeding levels over a 21-day period in the presence of SCF and GM-CSF. In lethally irradiated mice, survival enhancement and hematologic reconstitution were optimum with cells cultured for only seven days: survival at six months was 100% with cells cultured in SCF plus GM-CSF or SCF alone, compared to 50% with cells cultured with only GM-CSF. Hybridization analysis of bone marrow, spleen and thymus DNA from irradiated mice transplanted with these cultured cells confirmed male donor cell-derived repopulation at 45 days and 180 days post-transplant. These studies illustrate that murine GM-CFC can be expanded and that long-term repopulating hematopoietic cells can, at the minimum, be maintained ex vivo in serum-free culture. The use of defined serum-free culture systems holds great promise for further evaluation of the mechanisms that control hematopoietic stem cell proliferation.

Glycoprotein I identification during normal and pathological megakaryocytic maturation.

The expression of glycoprotein I (GP I) on normal and pathological megakaryocyte (MK) precursors has been investigated in vivo and in a cell culture system with mouse monoclonal antibody (AN51) using immunofluorescence, immunogold and immunoferritin with electron microscopy. Our results confirmed the observation of Rabellino et al. (1979, 1981), using polyclonal antibodies, that GP I was expressed throughout normal MK maturation. The present result differs from our previous work which failed to detect binding of AN51 on promegakaryoblasts (PMKB). Improvement of immunofluorescent techniques has permitted detection of weak fluorescent labeling on normal PMKB by day 6-7 of in vitro culture from normal CFU-MK. An increased number of labeled PMKB was observed in bone marrow from patients with idiopathic thrombocytopenic purpura (ITP) as compared with normal bone marrow. In 20 selected cases of acute megakaryoblastic leukemia, AN51 labeled the PMKB but at a lower percentage than with J15 (monoclonal anti-glycoprotein IIb/IIIa complex). The MK nature of the blasts was confirmed by the ultrastructural detection of platelet peroxidase (PPO) and by the binding of AN51 demonstrated with immunoferritin-conjugated anti-mouse IgG. The immunogold technique revealed that the density of gold particles on the cell membrane of PMKB was variable, but generally weaker than in platelets or pathological micromegakaryocytes. The specificity of AN51 for MK lineage was shown by the absence of binding of AN51, by all blasts from 60 cases of acute leukemia involving other cell lines. Because of its weak labeling of PMKB due to the small number of antigenic sites, AN51 must be associated with another megakaryocytic marker in the diagnostic assessment of acute megakaryoblastic leukemia.