The Effect of Perioperative Comprehensive Nursing on Patients Undergoing Ovary Endometriosis Laparoscopic Surgery.

Objective: To assess the effectiveness of perioperative nursing interventions in improving outcomes and satisfaction for patients undergoing laparoscopic surgery for ovarian endometriosis. Methods: From July 2021 to September 2022, 80 patients with endometriosis underwent laparoscopic surgery at Shijiazhuang Fourth Hospital and were randomly assigned to the conventional (n=40) and experimental (n=40) groups. During the perioperative period, patients in the conventional group received standard nursing interventions, while patients in the experimental group received comprehensive nursing interventions. The two groups were compared in terms of postoperative clinical indicators, self-rated anxiety scale (SAS) and self-rated depression scale (SDS) scores, nursing compliance, complications, and nursing satisfaction. Results: comprehensive nursing resulted in better postoperative clinical indices (time to get out of bed, hospital stay) versus routine nursing (all P < .001). The comprehensive nursing led to significantly lower SAS and SDS scores versus routine nursing. The nursing compliance of the patients in the experimental group was significantly higher than that of the patients in the conventional group (P < .001). Comprehensive nursing was associated with a significantly lower incidence of complications versus routine nursing (P < .001). Comprehensive nursing contributed to significantly higher nursing satisfaction versus routine nursing (P < .001). Conclusion: Comprehensive perioperative nursing interventions for patients with ovarian endometriosis undergoing laparoscopic surgery considerably accelerate patient recovery and enhance nursing compliance, as well as minimize patient negative emotions and improve patient satisfaction with nursing.  The comprehensive approach addresses the specific needs of patients during the recovery period, minimizing postoperative complications, accelerating patient recovery, and improving overall quality of life. By integrating psychological support, tailored strategies for pain management, early mobilization, and prompt intervention for complications, this intervention sets a benchmark for holistic care in gynecological surgery.

PpHY5 is involved in anthocyanin coloration in the peach flesh surrounding the stone.

Red coloration around the stone (Cs) is an important trait of canned peaches (Prunus persica). In this study, an elongated hypocotyl 5 gene in peach termed PpHY5 was identified to participate in the regulation of the Cs trait. The E3 ubiquitin ligase PpCOP1 was expressed in the flesh around the stone and could interact with PpHY5. Although HY5 is known to be degraded by COP1 in darkness, the PpHY5 gene was activated in the flesh tissue surrounding the stone at the ripening stages and its expression was consistent with anthocyanin accumulation. PpHY5 was able to promote the transcription of PpMYB10.1 through interacting with its partner PpBBX10. Silencing of PpHY5 in the flesh around the stone caused a reduction in anthocyanin pigmentation, while transient overexpression of PpHY5 and PpBBX10 resulted in anthocyanin accumulation in peach fruits. Moreover, transgenic Arabidopsis seedlings overexpressing PpHY5 showed increased anthocyanin accumulation in leaves. Our results improve our understanding of the mechanisms of anthocyanin coloration in plants.

PpHYH is responsible for light-induced anthocyanin accumulation in fruit peel of Prunus persica.

Peach Prunus persica is an economically important fruit tree crop worldwide. Although the external color of fruit is an important aspect of fruit quality, the mechanisms underlying its formation remain elusive in peach. Here, we report an elongated hypocotyl 5-homolog gene PpHYH involved in the regulation of anthocyanin pigmentation in peach fruit peel. Anthocyanin accumulation in fruit peel is light-dependent in peach. PpHYH had no auto-activation activity and its transcription was induced by sunlight. PpHYH activated transcription of a cluster of three PpMYB10 genes in the present of a cofactor PpBBX4 encoding a B-BOX protein, leading to anthocyanin accumulation in the sun-exposed peel. However, the PpHYH activity was repressed by a negative regulator of PpCOP1 encoding constitutive photomorphogenesis protein 1 which accumulated in the nucleus under dark condition, resulting in failure of anthocyanin accumulation in the shaded peel. PpCOP1 was re-localized into the cytosol under light condition, in accordance with fruit peel pigmentation. Additionally, transport of anthocyanins from the cytoplasm to the vacuole was a rate-limiting step for anthocyanin accumulation in peach fruit peel. Our results reveal for the first time the HYH gene involved in the regulation of anthocyanin accumulation in fruits, and provide target genes for genetic manipulation of fruit coloration.

Machine Learning Models for Predicting In-Hospital Mortality in Acute Aortic Dissection Patients.

Background: Acute aortic dissection is a potentially fatal cardiovascular disorder associated with high mortality. However, current predictive models show a limited ability to efficiently and flexibly detect this mortality risk, and have been unable to discover a relationship between the mortality rate and certain variables. Thus, this study takes an artificial intelligence approach, whereby clinical data-driven machine learning was utilized to predict the in-hospital mortality of acute aortic dissection. Methods: Patients diagnosed with acute aortic dissection between January 2015 to December 2018 were voluntarily enrolled from the Second Xiangya Hospital of Central South University in the study. The diagnosis was defined by magnetic resonance angiography or computed tomography angiography, with an onset time of the symptoms being within 14 days. The analytical variables included demographic characteristics, physical examination, symptoms, clinical condition, laboratory results, and treatment strategies. The machine learning algorithms included logistic regression, decision tree, K nearest neighbor, Gaussian naive bayes, and extreme gradient boost (XGBoost). Evaluation of the predictive performance of the models was mainly achieved using the area under the receiver operating characteristic curve. SHapley Additive exPlanation was also implemented to interpret the final prediction model. Results: A total of 1,344 acute aortic dissection patients were recruited, including 1,071 (79.7%) patients in the survivor group and 273 (20.3%) patients in non-survivor group. The extreme gradient boost model was found to be the most effective model with the greatest area under the receiver operating characteristic curve (0.927, 95% CI: 0.860-0.968). The three most significant aspects of the extreme gradient boost importance matrix plot were treatment, type of acute aortic dissection, and ischemia-modified albumin levels. In the SHapley Additive exPlanation summary plot, medical treatment, type A acute aortic dissection, and higher ischemia-modified albumin level were shown to increase the risk of hospital-based mortality.

Admission Systolic Blood Pressure and In-hospital Mortality in Acute Type A Aortic Dissection: A Retrospective Observational Study.

Background: Evidence between admission systolic blood pressure (SBP) and in-hospital deaths in acute type A aortic dissection (AAD) patients is inadequate. Here, we examined the relationship between SBP and in-hospital deaths in AAD patients. Methods: 703 AAD patients were enrolled from January 2014 to December 2018. The independent and dependent variables targeted were admission SBP and in-hospital deaths, respectively. Gender, age, body mass index (BMI), chronic renal insufficiency, smoking, hypertension, diabetes, laboratory indicators, and management were used as covariates. Results: The 703 participants had a mean age of 50.48 ± 11.35. About 76.24% of the participants were male. After adjusting for confounders, there was a negative correlation between AAD patients' admission SBP and in-hospital deaths (OR = 0.88, 95%CI 0.80-0.96). Consequently, a non-linear relationship of point 120 (mmHg) was detected between admission SBP and in-hospital deaths for AAD patients. Confidence intervals and effect sizes of the right (SBP >120 mmHg) and left (SBP ≤ 120 mmHg) sides of the inflection point were 0.96 (0.85-1.09) and 0.67 (0.51-0.88), respectively. The change in the male population and non-diabetes people was more pronounced according to subgroup analysis. Conclusions: Correlation between admission SBP and in-hospital mortality of AAD patients is non-linear. SBP negatively correlated with in-hospital mortality when ≤120 mmHg.

A candidate PpRPH gene of the D locus controlling fruit acidity in peach.

KEY MESSAGE: A candidate gene, designate PpRPH, in the D locus was identified to control fruit acidity in peach. Fruit acidity has a strong impact on organoleptic quality of fruit. Peach fruit acidity is controlled by a large-effect D locus on chromosome 5. In this study, the D locus was mapped to a 509-kb interval, with two markers, 5dC720 and 5C1019, co-segregating with the non-acid/acid trait of peach fruit. Within this interval, a candidate gene encoding a putative small protein, designated PpRPH, showed a consistency between gene expression and fruit acidity, with up- and down-regulation in non-acidic and acidic fruits, respectively. Transient ectopic expression of PpRPH in tobacco leaves caused an increase of pH by approximately 40% compared to the control transformed with empty vector. Whereas, the concentrations of citrate and malate decreased significantly by 22% and 37%, respectively, with respect to the empty vector control. All these results suggest that PpRPH is a strong candidate gene of the D locus. These findings contribute to our overall understanding of the complex mechanism underlying fruit acidity in peach as well as that in other fruit crops.

Characteristics and prognosis of acute type A aortic dissection with negative D-dimer result.

BACKGROUND: Evidence regarding the characteristics and prognosis in acute type A aortic dissection (AAD) patients with negative D-dimer result is limited. We aimed to investigate the characteristics and prognosis in AAD patients with negative D-dimer result. METHODS AND RESULTS: 370 AAD patients within 24 h of symptom onset were enrolled in a hospital in China from January 2014 to December 2018. Nine (2.43%) and 361 (97.57%) exhibited negative and positive D-dimer results, respectively. The average age of nine negative D-dimer result participants was 47.67 ± 10.95 years old, and about seven (77.78%) of them were male. The negative group showed a significantly lower blood pressure, white blood cell, hemoglobin, activated partial thromboplastin, ejection fraction and symptom with pain than the positive group. Multivariate analysis showed white blood cell (×109/L) (P = 0.008; odds ratio, 0.566) and symptom with pain (P < 0.001; odds ratio, 0.013) were significantly related to a negative result. The result of the fully-adjusted model showed negative D-dimer result was negatively associated with in-hospital mortality compared with positive group in AAD patients after adjusting confounders (OR = 0.34, 95%CI 0.01 to 10.82). CONCLUSIONS: Negative D-dimer result is strongly influenced by white blood cell and symptom with pain. Negative D-dimer result was negatively associated with in-hospital mortality compared with positive group in AAD patients.

Association between admission time and in-hospital mortality in acute aortic dissection patients: A retrospective cohort study.

PURPOSE: This study aimed to evaluate the relationship between admission time and in-hospital mortality in acute aortic dissection (AAD) patients. METHODS: The risk factors of in-hospital clinical outcomes were retrospectively evaluated in patients with AAD. All the patients were enrolled from January to December 2017 and were divided into two groups depending on the time of admission: daytime admissions were conducted from 8: 00 to 17: 30 hours whereas, nighttime admissions were from 17: 30 to 8: 00 hours. The primary endpoints were in-hospital mortality. Univariate and multivariable cox analyses were used to test the association between admission time and in-hospital mortality. RESULTS: The average age of the 363 participants in the present study was 52.25 ± 11.77 years, of which 81.6% were male. A total of 183 (50.4%) of these patients were admitted during nighttime. In-hospital mortality rate was higher in the nighttime admission group than in the daytime admission group (HR=1.86; 95%CI, 1.13 to 3.06, P=0.015). After adjusting for age, sex, and other risk factors, nighttime admission suggested as an independent risk factor for in-hospital mortality (HR=2.67, 95%CI, 1.30 to 5.46; P=0.007). Further subgroup analysis showed that none of the variables had a significant effect on the association between nighttime admission and in-hospital mortality. CONCLUSION: Nighttime admission for type A acute aortic dissection is associated with a higher risk of in-hospital mortality. Therefore, health care systems should focus on managing the increased risk of in-hospital mortality among patients admitted at night, regardless of the cause.

A nomogram for predicting in-hospital mortality in acute type A aortic dissection patients.

BACKGROUND: Although there are several biomarkers for identifying in-hospital mortality in acute type A aortic dissection (AAD), timely as well as perfect prediction in-hospital mortality is still not attained. Herein, we intend to develop as well to validate an in-hospital mortality risk independent predictive nomogram for AAD patients. METHODS: From January 2014 to December 2018, 703 individuals with AAD were involved in this study. They were indiscriminately categorized into training (n=520) and validation (n=183) sets. The univariate and multivariate analyses were used to screen in-hospital mortality predictors from the entire training set data. The predictors were used to establish a nomogram which was confirmed via internal as well as external authentication. This validation included discriminative capacity defined by the receiver operating characteristic (ROC) curve area under the curve (AUC) and the predictive precision via calibration curves. RESULTS: There was 33.43% in-hospital mortality overall incidence. The uric acid, D-dimer, C-reactive protein and management were individually related to in-hospital mortality as per multivariate logistic regression. On the basis of four variables with internal of AUC 0.901 and external validation of AUC 0.903, a nomogram was established. Calibration plots showed that the predicted and actual in-hospital mortality probabilities were fitted well on both internal and external validation. CONCLUSIONS: This recommended nomogram can calculate the specific possibility of in-hospital mortality with good precision, high discrimination, and probable clinical application in AAD patients.

Triglyceride/High-Density Lipoprotein Cholesterol Ratio Is Associated with In-Hospital Mortality in Acute Type B Aortic Dissection.

BACKGROUND: Triglyceride/high-density lipoprotein cholesterol (TG/HDL-c) ratio varies with vascular and other metabolic diseases. However, its role in acute type B aortic dissection is not well understood. In the current study, we evaluated the relationship between TG/HDL-c ratio and in-hospital mortality in type B aortic dissection. METHODS: We performed a retrospective analysis of consecutive patients between January 2015 and December 2018, by targeting dependent (TG/HDL-c ratio) and independent (in-hospital mortality) variables. TG/HDL-c ratio was determined as a division of TG levels by HDL-c levels. RESULTS: Of 523 patients in the study, we found a mean age of 55.00 ± 11.74 years, 15.68% of them being female. A fully-adjusted model revealed a positive relationship between TG/HDL-c ratio and in-hospital mortality in acute type B aortic dissection after adjusting confounders (OR = 2.08, 95% CI 1.32 to 3.27). This relationship was also nonlinear, with a point of 2.05. OR values (and confidence intervals) for the right (>2.05) and left (≤2.05) sides of the inflection point were 1.0 (0.580-1.26, P = 0.983) and 3.17 (1.54-6.57, P = 0.001), respectively. CONCLUSIONS: The TG/HDL-c ratio and in-hospital mortality in type B AAD have a nonlinear relationship among Chinese population. This ratio increased in-hospital mortality when it is less than 2.05.

Association of Lactate Dehydrogenase with In-Hospital Mortality in Patients with Acute Aortic Dissection: A Retrospective Observational Study.

BACKGROUND: Evidence regarding the relationship between serum lactate dehydrogenase (LDH) levels and in-hospital mortality in acute aortic dissection (AAD) patients is extremely limited. We aimed to investigate the relationship between LDH and in-hospital mortality in AAD patients. METHODS: The present study was a retrospective observational study. A total of 1526 participants with acute aortic dissection were involved in a hospital in China from January 2014 to December 2018. The target-independent variable was LDH measured at baseline, and the dependent was all-cause mortality during hospitalization. Covariates involved in this study included age, gender, body mass index (BMI), hypertension, diabetes, smoking, stroke, atherosclerosis, systolic blood pressure (SBP), diastolic blood pressure (DBP), white blood cell (WBC), hemoglobin (Hb), alanine transaminase (ALT), aspartate aminotransferase (AST), albumin (ALB), creatinine (Cr), symptom, type of AAD (Stanford), and management. RESULTS: The average age of 1526 selected participants was 52.72 ± 11.94 years old, and about 80.41% of them were male. The result of the fully adjusted model showed LDH was positively associated with in-hospital mortality in AAD patients after adjusting confounders (OR = 1.09, 95% CI 1.05 to 1.13). A nonlinear relationship was detected between LDH and in-hospital mortality in AAD patients after adjusting for potential confounders (age, gender, BMI, hypertension, diabetes, stroke, atherosclerosis, smoking, symptom, SBP, DBP, WBC, Hb, ALT, AST, ALB, Cr, type of AAD (Stanford), and management), whose point was 557. The effect sizes and the confidence intervals of the left and right sides of the inflection point were 0.90 (0.74-1.10) and 1.12 (1.06-1.19), respectively. Subgroup analysis in participants showed that the relationship between LDH and in-hospital mortality was stable, and all of the P value for the interaction in different subgroup were more than 0.05. CONCLUSIONS: The relationship between LDH and in-hospital mortality in AAD patients is nonlinear. LDH was positively related with in-hospital mortality when LDH is more than 557.

Ischemia-Modified Albumin, a Novel Predictive Marker of In-Hospital Mortality in Acute Aortic Dissection Patients.

BACKGROUND: This work explored the prognostic prediction capabilities of ischemia-modified albumin (IMA) in patients suffering from acute aortic dissection (AAD). METHODS: We conducted a retrospective analysis using electronic health records. This study included AAD patients admitted to the Second Xiangya Hospital of Central South University from January 2015 to December 2018 in ≤24 h from the onset of symptoms to hospital admission. The levels of IMA were recorded upon admittance and the final was the all-cause mortality during hospitalization. RESULTS: This study enrolled 731 AAD patients. Among who, 160 passed away in the course of medication while 571 of them survived. Those who passed away exhibited higher levels of IMA (94.35 ± 26.84 vs. 69.14 ± 14.70, p < 0.001) than the survivors. Following the adjustment confounders, the fully adjusted model showed IMA to be an independent forecastor for in-hospital mortality for AAD patients (OR 1.10, 95% CI 1.08-1.13, p < 0.001). Analysis based on receiver operating characteristic (ROC) revealed that 79.35 µ/ml was the best threshold of IMA level. The area under the curve (AUC) based on this IMA level was 0.854 (95% CI 0.822-0.898) while the specificity and sensitivity to anticipate in-hospital death were 84.8 and 80.6%, respectively. CONCLUSION: Admission IMA was an independent forecastor for in-hospital mortality among people suffering from AAD.

Activator-type R2R3-MYB genes induce a repressor-type R2R3-MYB gene to balance anthocyanin and proanthocyanidin accumulation.

Anthocyanin and proanthocyanidin (PA) accumulation is regulated by both myeloblastosis (MYB) activators and repressors, but little information is available on hierarchical interactions between the positive and negative regulators. Here, we report on a R2R3-MYB repressor in peach, designated PpMYB18, which acts as a negative regulator of anthocyanin and PA accumulation. PpMYB18 can be activated by both anthocyanin- and PA-related MYB activators, and is expressed both at fruit ripening and juvenile stages when anthocyanins or PAs, respectively, are being synthesized. The PpMYB18 protein competes with MYB activators for binding to basic Helix Loop Helixes (bHLHs), which develops a fine-tuning regulatory loop to balance PA and anthocyanin accumulation. In addition, the bHLH binding motif in the R3 domain and the C1 and C2 repression motifs in the C-terminus of PpMYB18 both confer repressive activity of PpMYB18. Our study also demonstrates a modifying negative feedback loop, which prevents cells from excess accumulation of anthocyanin and PAs, and serves as a model for balancing secondary metabolite accumulation at the transcriptional level.

Modulation of UVB-induced Carcinogenesis by Activation of Alternative DNA Repair Pathways.

The molecular basis for ultraviolet (UV) light-induced nonmelanoma and melanoma skin cancers centers on cumulative genomic instability caused by inefficient DNA repair of dipyrimidine photoproducts. Inefficient DNA repair and subsequent translesion replication past these DNA lesions generate distinct molecular signatures of tandem CC to TT and C to T transitions at dipyrimidine sites. Since previous efforts to develop experimental strategies to enhance the repair capacity of basal keratinocytes have been limited, we have engineered the N-terminally truncated form (Δ228) UV endonuclease (UVDE) from Schizosaccharomyces pombe to include a TAT cell-penetrating peptide sequence with or without a nuclear localization signal (NLS): UVDE-TAT and UVDE-NLS-TAT. Further, a NLS was engineered onto a pyrimidine dimer glycosylase from Paramecium bursaria chlorella virus-1 (cv-pdg-NLS). Purified enzymes were encapsulated into liposomes and topically delivered to the dorsal surface of SKH1 hairless mice in a UVB-induced carcinogenesis study. Total tumor burden was significantly reduced in mice receiving either UVDE-TAT or UVDE-NLS-TAT versus control empty liposomes and time to death was significantly reduced with the UVDE-NLS-TAT. These data suggest that efficient delivery of exogenous enzymes for the initiation of repair of UVB-induced DNA damage may protect from UVB induction of squamous and basal cell carcinomas.

Assessment of Sugar Components and Genes Involved in the Regulation of Sucrose Accumulation in Peach Fruit.

Soluble sugar contents in mature fruits of 45 peach accessions were quantified using gas chromatography analysis. Sucrose is the predominant sugar in mature fruit, followed by glucose and fructose, which have similar concentrations. Overall, sucrose metabolism and accumulation are crucial determinants of sugar content in peach fruit, and there is a wide range of sucrose concentrations among peach genotypes. To understand the mechanisms regulating sucrose accumulation in peach fruit, expression profiles of genes involved in sucrose metabolism and transport were compared among four genotypes. Two sucrose-cleaving enzyme genes (SUS4 and NINV8), one gene involved in sucrose resynthesis (SPS3), and three sugar transporter genes (SUT2, SUT4, and TMT2) were prevalently expressed in peach fruit, and their expression levels are significantly correlated with sucrose accumulation. In contrast, the VAINV genes responsible for sucrose cleavage in the vacuole were weakly expressed in mature fruit, suggesting that the sucrose-cleaving reaction is not active in the vacuole of sink cells of mature peach fruit. This study suggests that sucrose accumulation in peach fruit involves the coordinated interaction of genes related to sucrose cleavage, resynthesis, and transport, which could be helpful for future peach breeding.

Molecular genetics of blood-fleshed peach reveals activation of anthocyanin biosynthesis by NAC transcription factors.

Anthocyanin pigmentation is an important consumer trait in peach (Prunus persica). In this study, the genetic basis of the blood-flesh trait was investigated using the cultivar Dahongpao, which shows high levels of cyanidin-3-glucoside in the mesocarp. Elevation of anthocyanin levels in the flesh was correlated with the expression of an R2R3 MYB transcription factor, PpMYB10.1. However, PpMYB10.1 did not co-segregate with the blood-flesh trait. The blood-flesh trait was mapped to a 200-kb interval on peach linkage group (LG) 5. Within this interval, a gene encoding a NAC domain transcription factor (TF) was found to be highly up-regulated in blood-fleshed peaches when compared with non-red-fleshed peaches. This NAC TF, designated blood (BL), acts as a heterodimer with PpNAC1 which shows high levels of expression in fruit at late developmental stages. We show that the heterodimer of BL and PpNAC1 can activate the transcription of PpMYB10.1, resulting in anthocyanin pigmentation in tobacco. Furthermore, silencing the BL gene reduces anthocyanin pigmentation in blood-fleshed peaches. The transactivation activity of the BL-PpNAC1 heterodimer is repressed by a SQUAMOSA promoter-binding protein-like TF, PpSPL1. Low levels of PpMYB10.1 expression in fruit at early developmental stages is probably attributable to lower levels of expression of PpNAC1 plus the presence of high levels of repressors such as PpSPL1. We present a mechanism whereby BL is the key gene for the blood-flesh trait in peach via its activation of PpMYB10.1 in maturing fruit. Partner TFs such as basic helix-loop-helix proteins and NAC1 are required, as is the removal of transcriptional repressors.

Endothelin downregulates SERCA2 gene and protein expression in adult rat ventricular myocytes: regulation by pertussis toxin-sensitive Gi protein and cAMP.

Downregulation of the sarcoplasmic reticulum calcium ATPase (SERCA2) is associated with diastolic dysfunction in the failing heart. Elevated plasma endothelin-1 (ET) levels are correlated with congestive heart failure suggesting that ET may play a pathophysiological role. We have investigated the ability of ET to regulate SERCA2 gene expression in isolated adult rat ventricular myocytes. We find that ET enhances net protein synthesis by approximately 40% but significantly downregulates SERCA2 mRNA expression, time dependently, by approximately 30-50%, and the expression of SERCA2 protein by approximately 50%. In myoyctes, ET binds to ET(A) receptor that couples to G(q) and G(i) proteins. Inhibition of G(q)-PLC-induced phosphoinositide (PI) hydrolysis with U73122 (1 muM) or inhibition of G(i) protein with pertussis toxin (PTX) abolishes the ability of ET to downregulate SERCA2 mRNA gene expression. Further investigation suggests that ET coupling to PTX-sensitive G(i) with consequent lowering of cAMP is required for downregulation of SERCA2 mRNA levels. Increasing intracellular cAMP quantity using cAMP-specific PDE inhibitor Ro20-1724 or cAMP analog dibutyryl-cAMP reverses ET-induced downregulation of SERCA2 mRNA levels. The data indicate that, in adult myocytes, ET downregulates SERCA2 mRNA and protein levels, and the effect requires cross-talk between G(q) and PTX-sensitive G(i) pathways.

Transcriptionally active PCR for antigen identification and vaccine development: in vitro genome-wide screening and in vivo immunogenicity.

We have evaluated a technology called transcriptionally active PCR (TAP) for high throughput identification and prioritization of novel target antigens from genomic sequence data using the Plasmodium parasite, the causative agent of malaria, as a model. First, we adapted the TAP technology for the highly AT-rich Plasmodium genome, using well-characterized P. falciparum and P. yoelii antigens and a small panel of uncharacterized open reading frames from the P. falciparum genome sequence database. We demonstrated that TAP fragments encoding six well-characterized P. falciparum antigens and five well-characterized P. yoelii antigens could be amplified in an equivalent manner from both plasmid DNA and genomic DNA templates, and that uncharacterized open reading frames could also be amplified from genomic DNA template. Second, we showed that the in vitro expression of the TAP fragments was equivalent or superior to that of supercoiled plasmid DNA encoding the same antigen. Third, we evaluated the in vivo immunogenicity of TAP fragments encoding a subset of the model P. falciparum and P. yoelii antigens. We found that antigen-specific antibody and cellular immune responses induced by the TAP fragments in mice were equivalent or superior to those induced by the corresponding plasmid DNA vaccines. Finally, we developed and demonstrated proof-of-principle for an in vitro humoral immunoscreening assay for down-selection of novel target antigens. These data support the potential of a TAP approach for rapid high throughput functional screening and identification of potential candidate vaccine antigens from genomic sequence data.

Mutation of COOH-terminal lysines in overexpressed alpha B-crystallin abrogates ischemic protection in cardiomyocytes.

High levels of alpha B-crystallin are present in the cardiomyocyte, yet little is understood about the function and importance of this protein. Like many other small heat shock proteins, alpha B-crystallin forms large oligomeric complexes whose size can be regulated by posttranslational modifications. The size of these complexes can modify the function of the protein. A naturally occurring COOH-terminal mutant has many detrimental effects in the lens of the eye and altered oligomerization. Therefore, we mutated the two COOH-terminal lysines of alpha B-crystallin to glycines (K174/175G) and adenovirally mounted them to transduce cardiomyocytes. We analyzed the effect of this mutation on oligomerization, microtubular stabilization, and ischemic outcome. A nearly 45% downward shift in complex size was observed with the mutant by native PAGE followed by immunoblotting. The overexpressed protein no longer protected the tubulin cytoskeleton against ischemic stress by confocal analysis. The mutant caused a 30% increase in cytosolic enzyme release with ischemia compared with control, whereas a 33% decrease was associated with wild-type alpha B-crystallin overexpression. We conclude that the COOH terminus of alpha B-crystallin is crucial to its proper function.