Mammalian germ-line transgenesis by transposition

Transposons have been used in invertebrates for transgenesis and insertional mutagens in genetic screens. We tested a functional transposon called Sleeping Beauty in the one-cell mouse embryo. In this report, we describe experiments in which transposon vectors were injected into one-cell mouse embryos with mRNA expressing the SB10 transposase enzyme. Molecular evidence of transposition was obtained by cloning of insertion sites from multiple transgenic mice produced by SB10 mRNA/transposon coinjection. We also demonstrate germ-line transmission and expression from transposed elements. This technique has promise as a germ-line transgenesis method in other vertebrate species and for insertional mutagenesis in the mouse.     

Site-specific integrase-mediated transgenesis in mice via pronuclear injection

Microinjection of recombinant DNA into zygotic pronuclei has been widely used for producing transgenic mice. However, with this method, the insertion site, integrity, and copy number of the transgene cannot be controlled. Here, we present an integrase-based approach to produce transgenic mice via pronuclear injection, whereby an intact single-copy transgene can be inserted into predetermined chromosomal loci with high efficiency (up to 40%), and faithfully transmitted through generations. We show that neighboring transgenic elements and bacterial DNA within the transgene cause profound silencing and expression variability of the transgenic marker. Removal of these undesirable elements leads to global high-level marker expression from transgenes driven by a ubiquitous promoter. We also obtained faithful marker expression from a tissue-specific promoter. The technique presented here will greatly facilitate murine transgenesis and precise structure/function dissection of mammalian gene function and regulation in vivo.     

Site-specific recombination for the modification of transgenic strains of the Mediterranean fruit fly Ceratitis capitata

Insect transgenesis is mainly based on the random genomic integration of DNA fragments embedded into non-autonomous transposable elements. Once a random insertion into a specific location of the genome has been identified as particularly useful with respect to transgene expression, the ability to make the insertion homozygous, and lack of fitness costs, it may be advantageous to use that location for further modification. Here we describe an efficient method for the modification of previously inserted transgenes by the use of the site-specific integration system from phage phiC31 in a tephritid pest species, the Mediterranean fruit fly Ceratitis capitata. First, suitable transgenic strains with randomly integrated attP landing sites within transposon-based vectors were identified by molecular and functional characterization. Second, donor plasmids containing an attB site, with additional markers, and transposon ends were integrated into attP sites by phiC31 integrase-mediated recombination. Third, transposase-encoding ‘jumpstarter’ strains were created and mated to transgenic strains resulting in the postintegrational excision of transposon ends, which left stably integrated transgene insertions that could not be remobilized. This three-step integration and stabilization system will allow the combination of several transgene-encoded advantageous traits at evaluated genomic positions to generate optimized strains for pest control that minimize environmental concerns.     

Regulated transposition of a fish transposon in the mouse germ line

Tc1/mariner elements are able to transpose in species other than the host from which they were isolated. As potential vectors for insertional mutagenesis and transgenesis of the mouse, these cut-and-paste transposons were tested for their ability to transpose in the mouse germ line. First, the levels of activity of several Tc1/mariner elements in mammalian cells were compared; the reconstructed fish transposon Sleeping Beauty (SB) was found to be an order of magnitude more efficient than the other tested transposons. SB then was introduced into the mouse germ line as a two-component system: one transgene for the expression of the transposase in the male germ line and a second transgene carrying a modified transposon. In 20% of the progeny of double transgenic male mice the transposon had jumped from the original chromosomal position into another locus. Analysis of the integration sites shows that these jumps indeed occurred through the action of SB transposase, and that SB has a strong preference for intrachromosomal transposition. Analysis of the excision sites suggests that double-strand breaks in haploid spermatids are repaired via nonhomologous end joining. The SB system may be a powerful tool for transposon mutagenesis of the mouse germ line.     

Clinical significance of prognostic nutritional index in renal cell carcinomas

Prognostic nutritional index (PNI) could reflect the nutrition and inflammation status in cancer patients. This study aims to identify the prognostic significance of PNI in patients with renal cell carcinoma (RCC).A total of 694 RCC patients from our institution were included in this study. The prognostic correlation between PNI and overall survival (OS) and recurrence-free survival (RFS) was analyzed respectively using Kaplan–Meier method and univariate and multivariate Cox model. Studies about the association between pretreatment or preoperative PNI and prognosis of RCC were systemically reviewed and a meta-analysis method was performed to further evaluate the pooled prognostic value of PNI in RCC.267 (38.47%) RCC patients had low PNI according to the cut off value (49.08). Low PNI was associated with poor OS (P < .001) and RFS (P < .001), respectively. In the multivariate Cox analysis, PNI was identified to be an independent prognostic factor for OS (hazard ratio [HR] = 2.13, 95%CI: 1.25–3.62, P = .005). Compared to other nutritional indexes, this risk correlation of PNI is better than that of geriatric nutritional risk index (GNRI; HR = 1.19; P = .531), while is no better than that of neutrophil–lymphocyte ratio (NLR; 1/HR = 2.56; P < .001) and platelet–lymphocyte ratio (PLR; 1/HR = 2.85; P < .001) respectively. Meanwhile, additional 4785 patients from 6 studies were included into pooled analysis. For RCC patients who underwent surgery, low preoperative PNI was significantly associated with worse OS (pooled HR = 1.57, 95%CI: 1.37–1.80, P < .001) and worse RFS (pooled HR = 1.69, 95%CI: 1.45–1.96, P < .001). Furthermore, low PNI (<41–51) was also significantly associated with poor OS (HR = 1.78, 95%CI: 1.26–2.53 P < .05) and poor RFS (HR = 2.03, 95%CI: 1.40–2.95, P < .05) in advanced cases treated with targeted therapies.The present evidences show that PNI is an independent prognostic factor in RCC. Low PNI is significant associated with poor prognosis of RCC patients.     

Prognostic nutritional index predicts postoperative complications and long-term outcomes of gastric cancer

AIM:To investigate the impact of prognostic nutritional index(PNI)on the postoperative complications and long-term outcomes in gastric cancer patients undergoing total gastrectomy.METHODS:The data for 386 patients with gastric cancer were extracted and analyzed between January2003 and December 2008 in our center.The patients were divided into two groups according to the cutoff value of the PNI:those with a PNI≥46 and those with a PNI<46.Clinicopathological features were compared between the two groups and potential prognostic factors were analyzed.The relationship between postoperative complications and PNI was analyzed by logistic regression.The univariate and multivariate hazard ratios were calculated using the Cox proportional hazard model.RESULTS:The optimal cutoff value of the PNI was set at 46,and patients with a PNI≥46 and those with a PNI<46 were classified into PNI-high and PNI-low groups,respectively.Patients in the PNI-low group were more likely to have advanced tumor(T),node(N),and TNM stages than patients in the PNI-high group.The low PNI is an independent risk factor for the incidence of postoperative complications(OR=2.223).The 5-year overall survival(OS)rates were 54.1%and21.1%for patients with a PNI≥46 and those with a PNI<46,respectively.The OS rates were significantly lower in the PNI-low group than in the PNI-high group among patients with stagesⅡ(P=0.001)andⅢ(P<0.001)disease.CONCLUSION:The PNI is a simple and useful marker not only to identify patients at increased risk for postoperative complications,but also to predict long-term survival after total gastrectomy.The PNI should be included in the routine assessment of advanced gastric cancer patients.     

Is prognostic nutritional index a predictive marker for estimating all-cause in-hospital mortality in COVID-19 patients with cardiovascular risk factors?

BackgroundThis study examined the possible association between the prognostic nutritional index (PNI) and in-hospital mortality rates in cases with a high cardiovascular risk burden and hospitalized with the diagnosis of coronavirus disease 2019 (COVID-19).Material and MethodsThis retrospective and cross-sectional study included 294 COVID-19 patients hospitalized in a tertiary referral pandemic center. The study cohort was grouped into tertiles based on the initial PNI values as T1, T2, and T3. The PNI was calculated for each case and the prognostic value of this index was compared to CURB-65 and 4C mortality risk scores in predicting in-hospital mortality.ResultsPatients stratified into the T1 tertile had a lower lymphocyte count, serum albumin level, and PNI values. In a multivariate analysis, the PNI (OR: 0.688,%95CI: 0.586–0.808, p < 0.001) was an independent predictor for all-cause in-hospital death. After adjusting for confounding independent parameters, patients included in the T1 tertile were found to have 11.2 times higher rates of in-hospital mortality compared to the T3 group, which was presumed as the reference group. In addition, we found that the area under curve (AUC) value of PNI was significantly elevated than that of serum albumin level and total lymphocyte counts alone. [(AUC):0.79 vs AUC:0.75 vs AUC:0.69; respectively).ConclusionThis study demonstrated that the PNI is independently related with in-hospital mortality in patient with COVID-19 and cardiovascular risk factors. The power of the PNI was also validated using well-accepted risk scores of COVID-19 such as CURB-65 and 4C mortality risk scores.     

In vivo role of B lymphocytes in somatic transgene immunization

Immunity generated by in vivo inoculation of plasmid DNA is a straightforward and potentially valuable new approach to immunization. Little is known about the type of cells involved, the various immunological aspects, and the destiny of the transgene. In this report, we describe a system in which immunity is the result of in vivo targeting of B lymphocytes. This was accomplished using plasmid DNA encoding an immunoglobulin heavy-chain gene under the control of immunoglobulin promoter and enhancer elements. We show persistence of the transgene in splenic B lymphocytes for at least 3 months, i.e., the average life span of long-lived B lymphocytes in the mouse. The transgene could not be detected in any other lymphoid or nonlymphoid organs over a period of 6 months. We also established that the transgene is integrated in the host DNA. These studies bring new understanding to the events underlying the in vivo use of plasmid DNA. Moreover, the characteristics of this new approach make somatic transgene immunization a model system to study the immunogenicity of endogenous antigens in adult animals.     

A muscle-specific transgenic reporter line of the sea anemone,Nematostella vectensis

The sea anemone, Nematostella vectensis, has become an attractive new model organism for comparative genomics and evolutionary developmental biology. Over the last few years, many genes have been isolated and their expression patterns studied to gain insight into their function. More recently, functional tools have been developed to manipulate gene function; however, most of these approaches rely on microinjection and are limited to early stages of development. Transgenic lines would significantly enhance the tractability of the system. In particular, the study of gene- or tissue-specific promoters would be most useful. Here we report the stable establishment of a transgenic line using the I-SceI meganuclease system to facilitate integration into the genome. We isolated a 1.6-kb fragment of the regulatory upstream region of the Myosin Heavy Chain1 (MyHC1) gene and found that the transgene is specifically expressed in the retractor and tentacle muscles of Nematostella polyps, faithfully reproducing the expression of the endogenous MyHC1 gene. This demonstrates that the 1.6-kb fragment contains all of the regulatory elements necessary to drive correct expression and suggests that retractor and tentacle muscles in Nematostella are distinct from other myoepithelial cells. The transgene is transmitted through the germline at high frequency, and G₁ transgenic polyps have only one integration site. The relatively high frequency of transgenesis, in combination with gene- or tissue-specific promoters, will foster experimental possibilities for studying in vivo gene functions in gene regulatory networks and developmental processes in the nonbilaterian sea anemone, Nematostella vectensis.     

The association between the prognostic nutritional index and 28-day mortality among atrial fibrillation patients in China over 80 years of age

Backgrounds and aimsThe most prevalent form of cardiac rhythm abnormality among older populations is atrial fibrillation (AF). The prognostic nutritional index (PNI) is a reliable predictor of mortality in various diseases. The association between the PNI and mortality among AF patients over 80 years remains uncleared.Methods and resultsA retrospective assessment of AF cases admitted to a single cardiovascular disease unit in China between January 2015 and June 2020 was performed. The PNI at admission was defined as 10 × serum albumin (g/dL) + 0.005 × total lymphocyte count (per mm³). The association between PNI and cardiovascular disease (CVD)-related or all-cause mortality within 28 days was assessed via multivariable Cox regression. The analysis included 1141 patients. The CVD-related and all-cause mortality rates were 3.3% and 8.7%. Kaplan-Meier analyses revealed that cases with lower PNI tertiles exhibited higher all-cause mortality (T1: 7.6%; T2: 6.1%; T3: 2.4%, P < 0.001) or CVD mortality (T1: 6.3%; T2: 2.9%; T3: 0.8%, P < 0.001). After adjusting for potential confounders, continuous PNI was negatively related to the hazard of all-cause mortality (HR 0.92, 95% CI 0.89, 0.96) and CVD-related mortality (HR 0.90, 95% CI 0.84, 0.95). Compared to the T1 group, patients with a higher PNI exhibited a lower risk of all-cause mortality (P for trend 0.003) and CVD-related mortality (P for trend 0.005). Among most subgroups, CVD-related and all-cause mortality decreased with elevating PNI values.ConclusionsPNI is significantly negatively correlated with CVD-related and all-cause mortality among AF cases over 80 years.     

Efficient production by sperm-mediated gene transfer of human decay accelerating factor (hDAF) transgenic pigs for xenotransplantation

A large number of hDAF transgenic pigs to be used for xenotransplantation research were generated by using sperm-mediated gene transfer (SMGT). The efficiency of transgenesis obtained with SMGT was much greater than with any other method. In the experiments reported, up to 80% of pigs had the transgene integrated into the genome. Most of the pigs carrying the hDAF gene transcribed it in a stable manner (64%). The great majority of pigs that transcribed the gene expressed the protein (83%). The hDAF gene was transmitted to progeny. Expression was stable and found in caveolae as it is in human cells. The expressed gene was functional based on in vitro experiments performed on peripheral blood mononuclear cells. These results show that our SMGT approach to transgenesis provides an efficient procedure for studies involving large animal models.     

Novel immunological and nutritional-based prognostic index for gastric cancer

AIM: To assess the prognostic significance of immunological and nutritional-based indices, including the prognostic nutritional index(PNI), neutrophillymphocyte ratio(NLR), and platelet-lymphocyte ratio in gastric cancer.METHODS: We retrospectively reviewed 632 gastric cancer patients who underwent gastrectomy between1998 and 2008. Areas under the receiver operating characteristic curve were calculated to compare the predictive ability of the indices, together with estimating the sensitivity, specificity and agreement rate.Univariate and multivariate analyses were performed to identify risk factors for overall survival(OS). Propensity score analysis was performed to adjust variables to control for selection bias.RESULTS: Each index could predict OS in gastric cancer patients in univariate analysis, but only PNI had independent prognostic significance in multivariate analysis before and after adjustment with propensity scoring(hazard ratio, 1.668; 95% confidence interval:1.368-2.035). In subgroup analysis, a low PNI predicted a significantly shorter OS in patients with stage Ⅱ-Ⅲ disease(P = 0.019, P 0.001), T3-T4 tumors(P 0.001), or lymph node metastasis(P 0.001). Canton score, a combination of PNI, NLR, and platelet, was a better indicator for OS than PNI, with the largest area under the curve for 12-, 36-, 60-mo OS and overall OS(P = 0.022, P = 0.030, P 0.001, and P = 0.024,respectively). The maximum sensitivity, specificity, and agreement rate of Canton score for predicting prognosis were 84.6%, 34.9%, and 70.1%, respectively.CONCLUSION: PNI is an independent prognostic factor for OS in gastric cancer. Canton score can be a novel preoperative prognostic index in gastric cancer.     

Rapid postoperative reduction in prognostic nutrition index is associated with the development of pancreatic fistula following distal pancreatectomy

BackgroundsDespite recent advances in surgical techniques and devices for pancreatic remnant closure, postoperative pancreatic fistula (POPF) still remains one of the common complications after distal pancreatectomy (DP). Identification of risk factors for POPF may lead to the development of new strategies to prevent this ominous complication.MethodsWe retrospectively reviewed data on 44 patients undergoing DP with the use of a stapler to identify risk factors for POPF. Study variables included preoperative prognostic nutritional index (PNI) and reduction rate of PNI on postoperative day (POD) 7.ResultsPOPF occurred in 23 patients (52%), of which 13 (56%) were grade B or C. Univariate analyses comparing patients with POPF and those without POPF showed significant differences in body mass index (P = 0.0102), pancreatic thickness (P = 0.0134), white blood cell count on POD7 (P = 0.0432), C-reactive protein level on POD7 (P = 0.0123), and PNI reduction rate (P = 0.0471). A multivariate analysis revealed pancreatic thickness (P = 0.0121) and PNI reduction rate (P = 0.0165) to be significant factors for POPF. Furthermore, the PNI reduction rate was significantly higher in patients with clinically relevant (grade B/C) POPF than in those with no or grade A POPF (P = 0.0257). In most patients, the massive postoperative PNI reduction preceded the diagnosis of clinically relevant POPF.ConclusionsThese findings suggest that rapid postoperative reduction in PNI is associated with the development of POPF.     

Transgene- and locus-dependent imprinting reveals allele-specific chromosome conformations

When positioned into the integrin α-6 gene, an Hoxd9lacZ reporter transgene displayed parental imprinting in mouse embryos. While the expression from the paternal allele was comparable with patterns seen for the same transgene when present at the neighboring HoxD locus, almost no signal was scored at this integration site when the transgene was inherited from the mother, although the Itga6 locus itself is not imprinted. The transgene exhibited maternal allele-specific DNA hypermethylation acquired during oogenesis, and its expression silencing was reversible on passage through the male germ line. Histone modifications also corresponded to profiles described at known imprinted loci. Chromosome conformation analyses revealed distinct chromatin microarchitectures, with a more compact structure characterizing the maternally inherited repressed allele. Such genetic analyses of well-characterized transgene insertions associated with a de novo-induced parental imprint may help us understand the molecular determinants of imprinting.     

Prognostic nutritional index predicts in-hospital mortality in patients with acute type A aortic dissection

BackgroundThe prognostic nutritional index (PNI) has recently been reported to associate with the surgical prognosis of patients with some cardiovascular diseases. However, the prognosis significance of the preoperative PNI in patients with acute type A aortic dissection (AAAD) remains unclear.ObjectivesThe present study aimed to explore the relationship between PNI and postoperative in-hospital mortality in patients with AAAD.MethodsBetween June 2013 and December 2019, we retrospectively reviewed the clinical data of 651 patients undergoing AAAD surgery. Patients were divided into two groups according to the median PNI. The risk factors of postoperative in-hospital mortality were identified by univariate and multivariate logistic regression analysis.ResultsIn-hospital mortality was significantly more common in the low group (24.8% vs 16.3%: P = .007). The percentage of prolonged mechanical ventilation (58.9% vs 49.8%: P = .020) and the median duration of intensive care unit stays (7.0 vs 6.0 days: P = .003) were also higher and longer in the low group. Multivariate logistic regression analysis showed that the PNI, age, hypertension, and operation time independently predicted in-hospital mortality. Besides, compared with patients with a history of hypertension, the low PNI affected in-hospital mortality more than those without (odds ratio [OR]: 2.07; 95% confidence interval [CI]: 1.20-3.56; P = .009).ConclusionsLower PNI may be independently associated with in-hospital mortality of patients after AAAD surgery.     

Prognostic significance of baseline nutritional index for patients with esophageal squamous cell carcinoma after radical esophagectomy

Background

Radical esophagectomy is the cornerstone of curative treatment for patients with resectable esophageal squamous cell carcinoma (ESCC). Patient survival after surgery for ESCC is mainly associated with pathological tumor progression. Recently, the impact of baseline immune-nutritional status of various types of patients with cancer on survival has been highlighted. The purpose of the present study was to investigate the association between the baseline prognostic nutritional index (PNI) and postoperative short- and long-term results after esophagectomy for patients with ESCC.

Methods

In total, 202 patients with ESCC who underwent radical esophagectomy at our institution between 2002 and 2010 were enrolled. PNI was calculated as 10× serum albumin (g/dL) + 0.005 × total lymphocyte counts (per mm³). Receiver operating characteristic (ROC) curves were generated for multiple logistic regression analysis using 5-year overall survival as the endpoint to determine an optimal PNI cutoff value, in which patients were classified into two groups: high PNI and low PNI. We evaluated the significance of PNI on postoperative morbidity and long-term survival using univariate and multivariate analyses.

Results

The mean PNI was 48.9 ± 4.6 (range 37.2–64.0). The area under the ROC curve in multiple logistic regression analysis was 0.5367. The projected 5-year survival rate was optimal at a PNI of 44.1. Hence, the PNI cutoff point was set at 44, with subjects classified by PNI level into the low (PNI <44) or high (PNI ≥44) PNI groups. Of 202 patients, 173 (85.7 %) and 29 (14.3 %) were classified as having high and low PNI, respectively. No significant differences were noted between the two groups regarding patient background, including age, sex, pT, pN, and pStage, or postoperative complications. However, overall survival (OS) and relapse-free survival (RFS) were significantly worse in the low PNI group than in the high PNI group. The 5-year OS and RFS rates in the high PNI vs. low PNI groups were 67.2 vs. 41.2 % (P = 0.007) and 61.5 vs. 38.8 % (P = 0.008), respectively. Multivariate analysis revealed that PNI was a significant prognostic factor for both OS (hazard ratio, 1.826; 95 % confidence interval, 1.015–3.285; P = 0.044) and RFS (hazard ratio, 1.862; 95 % confidence interval, 1.121–3.095; P = 0.016).

Conclusion

Preoperative PNI is an independent prognostic marker of both OS and RFS for patients with potentially curative ESCC. A careful follow-up for tumor recurrence after surgery is required for ESCC patients with low PNI.

     

Prognostic nutritional index predicts outcomes of malignant pleural mesothelioma

Purpose

Nutritional status has been associated with long-time outcomes in cancer patients. We investigated whether the prognostic nutritional index (PNI), an indicator of nutritional status, affects overall survival in patients with malignant pleural mesothelioma (MPM).

Methods

We enrolled 121 patients with histologically confirmed MPM, who had successfully undergone biopsy by medical thoracoscopy in this study. Demographic, clinical and laboratory data were collected retrospectively. The PNI was calculated as 10× serum albumin value (g/dl) + 0.005 × total lymphocyte count (per mm³) in peripheral blood. Univariate and multivariate analyses were used to identify prognostic factors.

Results

Mean pretreatment PNI was 44.6. PNI was significantly associated with age (P = 0.031), smoking habits (P = 0.039) and weight loss (P = 0.029). Survival analysis showed PNI to be an independent prognostic factor in MPM. Patients with lower PNIs (PNI < 44.6) had greater risk of death than those with higher PNIs (PNI ≥ 44.6; hazard ratio: 2.290; 95 % confidence interval: 1.415–3.706; P = 0.001). These analyses were adjusted for patient age, gender, smoking habits, dyspnea, chest pain, weight loss, primary site of tumor, histology, platinum-based systemic chemotherapy, hospital and stage.

Conclusions

Pretreatment PNI is a novel independent prognostic factor in MPM.