A visual method for direct selection of high-producing Pichia pastoris clones

<p>Abstract</p> <p>Background</p> <p>The methylotrophic yeast, <it>Pichia pastoris</it>, offers the possibility to generate a high amount of recombinant proteins in a fast and easy way to use expression system. Being a single-celled microorganism, <it>P. pastoris </it>is easy to manipulate and grows rapidly on inexpensive media at high cell densities. A simple and direct method for the selection of high-producing clones can dramatically enhance the whole production process along with significant decrease in production costs.</p> <p>Results</p> <p>A visual method for rapid selection of high-producing clones based on mannanase reporter system was developed. The study explained that it was possible to use mannanase activity as a measure of the expression level of the protein of interest. High-producing target protein clones were directly selected based on the size of hydrolysis holes in the selected plate. As an example, the target gene (9elp-hal18) was expressed and purified in <it>Pichia pastoris </it>using this technology.</p> <p>Conclusions</p> <p>A novel methodology is proposed for obtaining the high-producing clones of proteins of interest, based on the mannanase reporter system. This system may be adapted to other microorganisms, such as <it>Saccharomyces cerevisiae </it>for the selection of clones.</p

A novel role of the splenic volume in Crohn’s disease: evaluating the efficacy of infliximab

Background: A number of patients with Crohn’s disease (CD) suffer from loss of response to infliximab (IFX) therapy. Splenic volume is reported to be enlarged in patients with CD compared to normal individuals. The association between splenic volume and IFX efficacy in CD remains unclear.Methods: We performed a retrospective study of patients with CD who received regular IFX treatment at Zhongshan Hospital, Fudan University, between August 2015 and December 2021. We collected baseline characteristics and clinical features from medical records in the CD database of Zhongshan Hospital. We accurately measured the splenic volume using semi-auto spleen segmentation software, followed by the analysis of splenic volume and IFX efficacy.Results: We included 49 patients with CD receiving IFX treatment, of whom 41 responded to IFX and 8 failed to respond to IFX. Splenic volume, as well as volume adjusted for body mass index (SV/BMI) and body weight (SV/W), was significantly decreased after IFX treatment in responders but increased in non-responders compared to the volume before the treatment. Accordingly, the levels of leukocyte count, platelet count, C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR) were decreased after IFX treatment in responders. Contrarily, the levels of hemoglobin, albumin, and tumor necrosis factor (TNF)-α were elevated in responders. Moreover, both CRP and TNF-α levels were significantly positively correlated with SV/BMI in all patients.Conclusion: Splenic volume, especially SV/BMI and SV/W, was reduced after IFX treatment in CD patients responsive to IFX. SV/BMI was positively correlated with disease activity. Splenic volume is a promising indicator to evaluate IFX efficacy in CD

CT volumetry can potentially predict the local stage for gastric cancer after chemotherapy

PURPOSE:We aimed to evaluate the value of CT tumor volumetry for predicting T and N stages of gastric cancer after chemotherapy, with pathologic results as the reference standard.METHODS:This study retrospectively evaluated 42 patients diagnosed with gastric cancer, who underwent chemotherapy followed by surgery. Pre- and post-treatment CT tumor volumes (VT) were measured in portal venous phase and volume reduction ratios were calculated. Correlations between pre- and post-treatment VT, reduction ratio, and pathologic stages were analyzed. Receiver operator characteristic (ROC) analyses were also performed to assess diagnostic performance for prediction of downstaging to T0–2 stage and N0 stage.RESULTS:Pretreatment VT, post-treatment VT, and VT reduction ratio were significantly correlated with T stage (rs=0.329, rs=0.546, rs= -0.422, respectively). Post-treatment VT and VT reduction ratio were significantly correlated with N stage (rs=0.442 and rs= -0.376, respectively). Pretreatment VT, post-treatment VT, and VT reduction ratio were significantly different between T0–2 and T3,4 stage tumors (P = 0.05, P < 0.001, and P = 0.002, respectively). The differences between N0 and ≥N1 groups were also statistically significant (P = 0.005 for post-treatment VT, P = 0.016 for VT reduction ratio, respectively). The area under the ROC curve (AUC) for identification of T0–2 groups was 0.70 for pretreatment VT, 0.88 for post-treatment VT, and 0.82 for VT reduction ratio, respectively. AUC was 0.78 for post-treatment VT and 0.74 for VT reduction ratio for identification of N0 groups.CONCLUSION:CT tumor volumetry, particularly post-treatment measurement of VT, is potentially valuable for predicting histopathologic T and N stages after chemotherapy in patients with gastric cancer

Magnetic resonance morphologic features predict progression of incidental pancreatic cystic lesions during follow-up

PURPOSEWe aimed to evaluate which morphologic features on magnetic resonance imaging (MRI) could predict the progression of pancreatic cystic lesions (PCLs) that are suitable for follow-up.METHODSA total of 2176 MRI findings of PCLs were retrospectively reviewed between January 2009 and December 2016. The study population was composed of 223 patients. Clinical data and morphologic features of PCLs were recorded. We divided the individuals into two sub-groups according to the final features on MRI. Univariable and multivariable regression analyses were performed to identify independent risk factors for progression of PCLs.RESULTSA total of 84 PCLs (37.7%) progressed during follow-up, while 139 PCLs (62.3%) were stable. Age (odds ratio [OR], 1.042; P = 0.017), number of lesions (OR, 0.491; P = 0.048), communication to pancreatic duct (PD) (OR, 2.425; P = 0.007) and presence of septa (OR, 6.105; P < 0.001) were significant independent factors for progression of PCLs. Among 84 lesions that progressed, 23 lesions (27.4%) increased to ≥ 30 mm in diameter or showed worrisome imaging features at the end of follow-up that needed clinical intervention. The initial size and communication to PD were independent factors for progression of PCLs necessitating clinical intervention (P < 0.001 and P = 0.011, respectively).CONCLUSIONAge, number of the lesions, communication to PD and presence of septa were independent risk factors for the progression of PCLs, and the initial size and communication to PD could potentially predict PCLs needing clinical intervention

MR quantitative 3D shape analysis helps to distinguish mucinous cystic neoplasm from serous oligocystic adenoma

PURPOSEWe aimed to assess the performance of quantitative 3D shape analysis in the differential diagno- sis of pancreatic serous oligocystic adenoma (SOA) and mucinous cystic neoplasm (MCN).METHODSFour hundred thirty-two patients diagnosed with serous cystic neoplasms (SCNs) or MCNs were retrospectively reviewed from August 2014 to July 2019 and finally 87 patients with MCNs (n = 45) and SOAs (n = 42) were included. Clinical data and magnetic resonance morphologic fea- tures with 3D shape analysis of lesions (shape sphericity, compacity, and volume) were recorded and compared between MCNs and SOAs according to the pathology. Univariable and multivari- able regression analyses were used to identify independent impact factors for differentiating MCN from SOA.RESULTSThe age of MCN patients was younger than SOAs (43.02 ± 10.83 years vs. 52.78 ± 12.31 years; OR = 0.275; 95% CI: 0.098-0.768; P = .014). MCN has a higher female/male ratio than SOA (43/2 vs. 27/15; OR = 40.418; 95% CI: 2.704-604.171; P = .007) and was more often located in the distal of pancreas (OR = 31.403; 95% CI: 2.985-330.342; P = .004). Shape_Sphericity derived from 3D shape analysis was a significant independent factor in the multivariable analysis and the value of MCN was closer to 1 than SOA (OR = 35.153; 95% CI: 5.301-237.585; P < .001). Area under the receiver operating characteristic curve (AUC) of Shape_Sphericity was 0.923 (optimal cutoff value was 0.964876).CONCLUSIONShape_Sphericity in combination with age, sex, and location could help to distinguish MCN from SOA

Value of apparent diffusion coefficient for predicting malignancy of intraductal papillary mucinous neoplasms of the pancreas

PURPOSE: We aimed to explore the potential value of the whole tumor apparent diffusion coefficient (ADC) for discriminating between benign and malignant intraductal papillary mucinous neoplasms (IPMNs) of the pancreas.METHODS:Forty-two patients underwent 1.5 T magnetic resonance imaging that included diffusion-weighted imaging (DWI, b=0.500 s/mm2). The mean, minimum, and maximum ADC values were measured for the whole tumor. The differences between benign and malignant IPMNs were calculated for the mean ADC, ADC-min, and ADC-max values. Receiver operating characteristics (ROC) analysis was conducted to evaluate their potential diagnostic performance.RESULTS:Fifteen of 25 benign IPMNs demonstrated low or iso-signal intensity on DWI with a b value of 500 s/mm2 compared with normal pancreatic parenchyma, whereas all malignant IPMNs demonstrated high signal intensity. The mean value of ADC was significantly higher in benign IPMNs compared with malignant IPMNs (3.39×10−3 mm2/s vs. 2.39×10−3 mm2/s, P < 0.001), with an area under the ROC curve (AUC) of 0.92 (95% confidence interval [CI], 0.79–0.98). The ADC-min value of malignant IPMNs was also significantly lower than that of benign IPMNs (1.24×10−3 mm2/s vs. 2.58×10−3 mm2/s, P < 0.001), with an AUC of 0.94 (95% CI, 0.82–0.99). No marked difference was found between benign and malignant IPMNs for the ADC-max value (3.89×10−3 mm2/s vs. 3.78×10−3 mm2/s, P = 0.299).CONCLUSION:Lower mean and minimum ADC values of the whole tumor might be potential predictors of malignant IPMNs of the pancreas

Feasibility of histogram analysis of susceptibility-weighted MRI for staging of liver fibrosis

PURPOSE:We aimed to evaluate whether histogram analysis of susceptibility-weighted imaging (SWI) could quantify liver fibrosis grade in patients with chronic liver disease (CLD).METHODS:Fifty-three patients with CLD who underwent multi-echo SWI (TEs of 2.5, 5, and 10 ms) were included. Histogram analysis of SWI images were performed and mean, variance, skewness, kurtosis, and the 1st, 10th, 50th, 90th, and 99th percentiles were derived. Quantitative histogram parameters were compared. For significant parameters, further receiver operating characteristic (ROC) analyses were performed to evaluate the potential diagnostic performance for differentiating liver fibrosis stages.RESULTS:The number of patients in each pathologic fibrosis grade was 7, 3, 5, 5, and 33 for F0, F1, F2, F3, and F4, respectively. The results of variance (TE: 10 ms), 90th percentile (TE: 10 ms), and 99th percentile (TE: 10 and 5 ms) in F0–F3 group were significantly lower than in F4 group, with areas under the ROC curves (AUCs) of 0.84 for variance and 0.70–0.73 for the 90th and 99th percentiles, respectively. The results of variance (TE: 10 and 5 ms), 99th percentile (TE: 10 ms), and skewness (TE: 2.5 and 5 ms) in F0–F2 group were smaller than those of F3/F4 group, with AUCs of 0.88 and 0.69 for variance (TE: 10 and 5 ms, respectively), 0.68 for 99th percentile (TE: 10 ms), and 0.73 and 0.68 for skewness (TE: 2.5 and 5 ms, respectively).CONCLUSION:Magnetic resonance histogram analysis of SWI, particularly the variance, is promising for predicting advanced liver fibrosis and cirrhosis

Comparison of diffusion-weighted MRI acquisition techniques for normal pancreas at 3.0 Tesla

PURPOSEWe aimed to optimize diffusion-weighted imaging (DWI) acquisitions for normal pancreas at 3.0 Tesla.MATERIALS AND METHODSThirty healthy volunteers were examined using four DWI acquisition techniques with b values of 0 and 600 s/mm2 at 3.0 Tesla, including breath-hold DWI, respiratory-triggered DWI, respiratory-triggered DWI with inversion recovery (IR), and free-breathing DWI with IR. Artifacts, signal-to-noise ratio (SNR) and apparent diffusion coefficient (ADC) of normal pancreas were statistically evaluated among different DWI acquisitions.RESULTSStatistical differences were noticed in artifacts, SNR, and ADC values of normal pancreas among different DWI acquisitions by ANOVA (P < 0.001). Normal pancreas imaging had the lowest artifact in respiratory-triggered DWI with IR, the highest SNR in respiratory-triggered DWI, and the highest ADC value in free-breathing DWI with IR. The head, body, and tail of normal pancreas had statistically different ADC values on each DWI acquisition by ANOVA (P < 0.05).CONCLUSIONThe highest image quality for normal pancreas was obtained using respiratory-triggered DWI with IR. Normal pancreas displayed inhomogeneous ADC values along the head, body, and tail structures

Cytokine-induced killer cells efficiently kill stem-like cancer cells of nasopharyngeal carcinoma via the NKG2D-ligands recognition

Cancer stem cells (CSCs) are considered to be the root cause for cancer treatment failure. Thus, there remains an urgent need for more potent and safer therapies against CSCs for curing cancer. In this study, the antitumor activity of cytokine-induced killer (CIK) cells against putative CSCs of nasopharyngeal carcinoma (NPC) was fully evaluated in vitro and in vivo. To visualize putative CSCs in vitro by fluorescence imaging, and image and quantify putative CSCs in tumor xenograft-bearing mice by in vivo bioluminescence imaging, NPC cells were engineered with CSC detector vector encoding GFP and luciferase (Luc) under control of Nanog promoter. Our study reported in vitro intense tumor-killing activity of CIK cells against putative CSCs of NPC, as revealed by percentage analysis of side population cells, tumorsphere formation assay and Nanog-promoter-GFP-Luc reporter gene strategy plus time-lapse recording. Additionally, time-lapse imaging firstly illustrated that GFP-labeled or PKH26-labeled putative CSCs or tumorspheres were usually attacked simultaneously by many CIK cells and finally killed by CIK cells, suggesting the necessity of achieving sufficient effector-to-target ratios. We firstly confirmed that NKG2D blockade by anti-NKG2D antibody significantly but partially abrogated CIK cell-mediated cytolysis against putative CSCs. More importantly, intravenous infusion of CIK cells significantly delayed tumor growth in NOD/SCID mice, accompanied by a remarkable reduction in putative CSC number monitored by whole-body bioluminescence imaging. Taken together, our findings suggest that CIK cells demonstrate the intense tumor-killing activity against putative CSCs of NPC, at least in part, by NKG2D-ligands recognition. These results indicate that CIK cell-based therapeutic strategy against CSCs presents a promising and safe approach for cancer treatment