Bispecific antibodies, which can bind to two different epitopes on the same or different antigens simultaneously, have recently emerged as attractive candidates for study in various diseases. Our ...present study successfully constructs and expresses a fully human, bispecific, single-chain diabody (BsDb) that can bind to vascular endothelial growth factor 165 (VEGF165) and programmed death-1 (PD-1) in
. Under the optimal expression conditions (methanol concentration, 1%; pH, 4.0; inoculum density, OD600 = 4, and the induction time, 96 h), the maximum production level of this BsDb is achieved at approximately 20 mg/L. The recombinant BsDb is purified in one step using nickel-nitrilotriacetic acid (Ni-NTA) column chromatography with a purity of more than 95%. Indirect enzyme-linked immune sorbent assay (ELISA) and sandwich ELISA analyses show that purified BsDb can bind specifically to VEGF165 and PD-1 simultaneously with affinities of 124.78 nM and 25.07 nM, respectively. Additionally, the BsDb not only effectively inhibits VEGF165-stimulated proliferation, migration, and tube formation in primary human umbilical vein endothelial cells (HUVECs), but also significantly improves proliferation and INF-γ production of activated T cells by blocking PD-1/PD-L1 co-stimulation. Furthermore, the BsDb displays potent antitumor activity in mice bearing HT29 xenograft tumors by inhibiting tumor angiogenesis and activating immune responses in the tumor microenvironment. Based on these results, we have prepared a potential bispecific antibody drug that can co-target both VEGF165 and PD-1 for the first time. This work provides a stable foundation for the development of new strategies by the combination of an angiogenesis inhibition and immune checkpoint blockade for cancer therapy.
The classical and alternative activation of macrophages has been proposed to play a role in radiation-induced pneumonitis and fibrosis, respectively. To test this hypothesis, the thoraces of C57BL/6 ...mice were irradiated with 12 Gy X-rays, and irradiated and control mice were euthanized at 1, 8, 12, 24 and 72 hours, and 2, 4, 8, 16 and 24 weeks after irradiation. The expression of inducible nitric oxide synthase (iNOS) and arginase type 1 (Arg-1) was evaluated at the mRNA and protein levels at different stages post-irradiation. We demonstrated that the enhanced mRNA and protein expression of iNOS occurred within the pneumonic stage, whereas the high levels of Arg-1 expression occurred within the fibrotic phase. Immunohistochemistry revealed that iNOS and Arg-1 were mainly expressed in macrophages. The expression of iNOS and Arg-1 may be associated with acute radiation pneumonitis and the development of radiation fibrosis, respectively. Although the function of macrophages cannot explain the whole process of radiation-induced pulmonary injury development, it may play an important regulatory role during this process.
To evaluate the contribution of chemotherapy for patients with locoregionally advanced nasopharyngeal carcinoma (NPC) treated by intensity modulated radiotherapy (IMRT) and to identify the optimal ...combination treatment strategy.
Between 2006 and 2010, 276 patients with stage II-IVb NPC were treated by IMRT alone or IMRT plus chemotherapy. Cisplatin-based chemotherapy included neoadjuvant or concurrent, or neoadjuvant plus concurrent protocols. The IMRT alone and chemoradiotherapy groups were well-matched for prognostic factors, except N stage, with more advanced NPC in the chemoradiotherapy arm.
With a mean follow-up of 33.8 months, the 3-year actuarial rates of overall survival (OS), metastasis-free survival (MFS), relapse-free survival (RFS), and disease-free survival (DFS) were 90.3%, 84.2%, 80.3%, and 69.2% for all of the patients, respectively. Compared with the IMRT alone arm, patients treated by concurrent chemoradiotherapy had a significantly better DFS (HR = 2.64; 95% CI, 1.12-6.22; P = 0.03), patients with neoadjuvant-concurrent chemoradiotherapy had a significant improvement in RFS and DFS (HR = 4.03; 95% CI, 1.35-12.05; P = 0.01 and HR = 2.43; 95% CI, 1.09-5.44; P = 0.03), neoadjuvant chemoradiotherapy provided no significant benefit in OS, MFS, RFS, and DFS. Stage group and alcohol consumption were prognostic factors for OS and N stage was a significant predictor for DFS.
Addition of concurrent or neoadjuvant-concurrent chemotherapy to IMRT is available to prolong RFS or DFS for locoregionally advanced NPC. Such work could be helpful to guide effective individualized therapy.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
ABSTRACT
Reproductive stage water stress leads to spikelet sterility in wheat. Whereas drought stress at anthesis affects mainly grain size, stress at the young microspore stage of pollen development ...is characterized by abortion of pollen development and reduction in grain number. We identified genetic variability for drought tolerance at the reproductive stage. Drought‐tolerant wheat germplasm is able to maintain carbohydrate accumulation in the reproductive organs throughout the stress treatment. Starch depletion in the ovary of drought‐sensitive wheat is reversible upon re‐watering and cross‐pollination experiments indicate that the ovary is more resilient than the anther. The effect on anthers and pollen fertility is irreversible, suggesting that pollen sterility is the main cause of grain loss during drought conditions in wheat. The difference in storage carbohydrate accumulation in drought‐sensitive and drought‐tolerant wheat is correlated with differences in sugar profiles, cell wall invertase gene expression and expression of fructan biosynthesis genes in anther and ovary (sucrose : sucrose 1‐fructosyl‐transferase, 1‐SST; sucrose : fructan 6‐fructosyl‐transferase, 6‐SFT). Our results indicate that the ability to control and maintain sink strength and carbohydrate supply to anthers may be the key to maintaining pollen fertility and grain number in wheat and this mechanism may also provide protection against other abiotic stresses.
Invertases catalyze the irreversible hydrolysis of sucrose to glucose and fructose. Plants contain two unrelated families of these enzymes: acid forms that derive from periplasmic invertases of ...eubacteria and are found in cell wall and vacuole, and neutral/alkaline forms evolved from the cytosolic invertases of cyanobacteria. Genomes of rice (Oryza sativa) and thale cress (Arabidopsis thaliana) contain multiple genes encoding these two families. Here for rice we identify the member genes of a cell-wall group (designated OsCIN1-9), a vacuolar group (OsVIN1-2), and two ancient neutral/alkaline groups: alpha (OsNIN1-4) and beta (OsNIN5-8). In Arabidopsis these groups contain six, two, four and five members, respectively. It is believed that the vacuolar group evolved from the cell-wall group. We provide evidence that the N-terminal signal peptide that directs cell-wall invertases co-translationally into the endoplasmic reticulum for secretion was replaced in the vacuolar group by a sequence similar to the complex N-terminal motif that targets alkaline phosphatase post-translationally to the vacuolar membrane of yeast. Since the last common ancestor of Arabidopsis and rice, the two invertase families evolved equally rapidly via gene duplication and gene loss, but the acid invertase family underwent approximately 10 events of intron loss compared with a single event of intron gain in the neutral/alkaline invertase family. Transcripts were detected for all rice invertase genes except OsCIN9. The acid invertase genes showed greater spatial and temporal diversity of expression than the neutral/alkaline genes.
High mobility group box 1(HMGB1) was first recognized as a nuclear protein that increased the chromatin remodeling and regulates transcription of many genes. In recent years, HMGB1 has been ...identified as a critical "late" pro-inflammatory mediator due to its unique secretion pattern and lethal effects in sepsis. Therefore, preventing the active release and inhibiting the pro-inflammatory activity of HMGB1 become promising strategies for the treatment of sepsis. Here, we reported the therapeutic effects of Gu-4, a lactosyl derivative, on sepsis and the underlying molecular mechanisms.
In an experimental rat model of sepsis caused by cecal ligation and puncture (CLP), Gu-4 administration prominently attenuated lung injury and improved the survival of the septic animals, which was positively correlated with the decrease of the serum HMGB1 level. Using RAW264.7 macrophage cell line, we further showed that Gu-4 significantly suppressed the lipopolysaccharide (LPS)-induced release and cytoplasmic translocation of HMGB1. Moreover, Gu-4 not only dose-dependently attenuated recombinant human (rhHMGB1)-induced production of TNF-α, IL-6, and IL-1β in THP-1 cells, but also greatly inhibited the adhesion of rhHMGB1-challenged THP-1 cells to HUVECs. Analyses of flow cytometry demonstrated that Gu-4 could effectively reduce the activation of CD11b elicited by rhHMGB1. Western blot analyses revealed that Gu-4 treatment could partially block the rhHMGB1-induced activation of ERK and NF-κB signalings. Meanwhile, CD11b knockdown also obviously attenuated the rhHMGB1-induced phosphorylations of ERK and IKKα/β.
Taken together, our results suggest that Gu-4 possesses a therapeutic potential in the treatment of sepsis probably via inhibiting the LPS-induced release of HMGB1 from macrophages and via suppressing the pro-inflammatory activity of HMGB1.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The 15q25.1 lung cancer susceptibility locus, containing CHRNA5, could modify lung cancer susceptibility and multiple smoking related phenotypes. However, no studies have investigated the association ...between CHRNA5 rs3841324, which has been proven to have the highest association with CHRNA5 mRNA expression, and the risk of other smoking-associated cancers, except lung cancer. In the current study we examined the association between rs3841324 and susceptibility to smoking-associated nasopharyngeal carcinoma (NPC).
In this case-control study we genotyped the CHRNA5 rs3841324 polymorphism with 400 NPC cases and 491 healthy controls who were Han Chinese and frequency-matched by age (±5 years), gender, and alcohol consumption. Univariate and multivariate logistic regression analyses were used to calculate the odds ratio (OR) and 95% confidence intervals (95% CI).
We found that individuals with CHRNA5 rs3841324 combined variant genotypes (ins/del+del/del) had a >1.5-fold elevated risk for NPC than those with the ins/ins genotype (adjusted OR = 1.52; 95% CI, 1.16-2.00), especially among ever smokers (adjusted OR = 2.07; 95% CI, 1.23-3.48). The combined variant genotypes acted jointly with cigarette smoking to contribute to a 4.35-fold increased NPC risk (adjusted OR = 4.35; 95% CI, 2.57-7.38). There was a dose-response relationship between deletion alleles and NPC susceptibility (trend test, P = 0.011).
Our results suggest that genetic variants on the 15q25.1 lung cancer susceptibility locus may influence susceptibility to NPC, particularly for smoking-associated NPC. Such work may be helpful to facilitate an understanding of the etiology of smoking-associated cancers and improve prevention efforts.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
As central components of RNA silencing, small RNAs play diverse and important roles in many biological processes in eukaryotes. Aberrant reduction or elevation in the levels of small RNAs is ...associated with many developmental and physiological defects. The in vivo levels of small RNAs are precisely regulated through modulating the rates of their biogenesis and turnover. 2＇-O-methylation on the 3＇ terminal ribose is a major mechanism that increases the stability of small RNAs. The small RNA methyltransferase HUA ENHANCER1 （HEN1） and its homologs methylate microRNAs and small interfering RNAs （siRNAs） in plants, Piwi-interacting RNAs （piRNAs） in animals, and siRNAs in Drosophila. 3＇ nucleotide addition, especially uridylation, and 3＇-5＇ exonucleolytic degradation are major mechanisms that turnover small RNAs. Other mechanisms impacting small RNA stability include complementary RNAs, cis-elements in small RNA sequences and RNA-binding proteins. Investigations are ongoing to further understand how small RNA stability impacts their accumulation in vivo in order to improve the utilization of RNA silencing in biotechnology and therapeutic applications.
Genome-wide association studies (GWAS) have proven successful in predicting genetic risk of disease using single-locus models; however, identifying single nucleotide polymorphism (SNP) interactions ...at the genome-wide scale is limited due to computational and statistical challenges. We addressed the computational burden encountered when detecting SNP interactions for survival analysis, such as age of disease-onset. To confront this problem, we developed a novel algorithm, called the Efficient Survival Multifactor Dimensionality Reduction (ES-MDR) method, which used Martingale Residuals as the outcome parameter to estimate survival outcomes, and implemented the Quantitative Multifactor Dimensionality Reduction method to identify significant interactions associated with age of disease-onset.
To demonstrate efficacy, we evaluated this method on two simulation data sets to estimate the type I error rate and power. Simulations showed that ES-MDR identified interactions using less computational workload and allowed for adjustment of covariates. We applied ES-MDR on the OncoArray-TRICL Consortium data with 14,935 cases and 12,787 controls for lung cancer (SNPs = 108,254) to search over all two-way interactions to identify genetic interactions associated with lung cancer age-of-onset. We tested the best model in an independent data set from the OncoArray-TRICL data.
Our experiment on the OncoArray-TRICL data identified many one-way and two-way models with a single-base deletion in the noncoding region of BRCA1 (HR 1.24, P = 3.15 × 10
), as the top marker to predict age of lung cancer onset.
From the results of our extensive simulations and analysis of a large GWAS study, we demonstrated that our method is an efficient algorithm that identified genetic interactions to include in our models to predict survival outcomes.
• Fructans are believed to contribute to cold and drought tolerance in several plant families (Poaceae, Asparagaceae and Asteraceae), but it is not clear why the ability to accumulate these polymers ...is found in some genera (e.g. Triticum) but not in others (e.g. Oryza). • As fructan biosynthesis enzymes (FBEs) evolved from vacuolar invertases (VINs), we searched the rice genome sequence for genes related to both FBE and VIN genes of wheat and other members of the Pooideae. We compared them at the levels of exon-intron structure, protein sequence, and the enzymatic properties of recombinant proteins after expression in the yeast Pichia pastoris. • We found that rice possesses two VIN genes (OsVIN1 and OsVIN2) and no FBE genes. FBE genes appear to have arisen in the Pooideae by a series of gene duplications from an ancestor of wheat TaVIN3. Recombinant TaVIN2, OsVIN1 and OsVIN2 behaved as invertases with no FBE activity, but possessed high fructan exohydrolase activity, especially OsVIN1. • The engineering of fructan accumulation into rice for greater stress tolerance could founder on endogenous exohydrolases, but the fact that OsVIN1 transcripts are absent from peduncles of well watered and drought-stressed plants removes one potential obstacle to this endeavour.
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