Delayed mortality among solid organ transplant recipients hospitalized for COVID-19.

INTRODUCTION: Most studies of solid organ transplant (SOT) recipients with COVID-19 focus on outcomes within one month of illness onset. Delayed mortality in SOT recipients hospitalized for COVID-19 has not been fully examined. METHODS: We used data from a multicenter registry to calculate mortality by 90 days following initial SARS-CoV-2 detection in SOT recipients hospitalized for COVID-19 and developed multivariable Cox proportional-hazards models to compare risk factors for death by days 28 and 90. RESULTS: Vital status at day 90 was available for 936 of 1117 (84%) SOT recipients hospitalized for COVID-19: 190 of 936 (20%) died by 28 days and an additional 56 of 246 deaths (23%) occurred between days 29 and 90. Factors associated with mortality by day 90 included: age > 65 years [aHR 1.8 (1.3-2.4), p =<0.001], lung transplant (vs. non-lung transplant) [aHR 1.5 (1.0-2.3), p=0.05], heart failure [aHR 1.9 (1.2-2.9), p=0.006], chronic lung disease [aHR 2.3 (1.5-3.6), p<0.001] and body mass index ≥ 30 kg/m 2 [aHR 1.5 (1.1-2.0), p=0.02]. These associations were similar for mortality by day 28. Compared to diagnosis during early 2020 (March 1-June 19, 2020), diagnosis during late 2020 (June 20-December 31, 2020) was associated with lower mortality by day 28 [aHR 0.7 (0.5-1.0, p=0.04] but not by day 90 [aHR 0.9 (0.7-1.3), p=0.61]. CONCLUSIONS: In SOT recipients hospitalized for COVID-19, >20% of deaths occurred between 28 and 90 days following SARS-CoV-2 diagnosis. Future investigations should consider extending follow-up duration to 90 days for more complete mortality assessment.

Changing trends in mortality among solid organ transplant recipients hospitalized for COVID-19 during the course of the pandemic.

Mortality among patients hospitalized for COVID-19 has declined over the course of the pandemic. Mortality trends specifically in solid organ transplant recipients (SOTR) are unknown. Using data from a multicenter registry of SOTR hospitalized for COVID-19, we compared 28-day mortality between early 2020 (March 1, 2020-June 19, 2020) and late 2020 (June 20, 2020-December 31, 2020). Multivariable logistic regression was used to assess comorbidity-adjusted mortality. Time period of diagnosis was available for 1435/1616 (88.8%) SOTR and 971/1435 (67.7%) were hospitalized: 571/753 (75.8%) in early 2020 and 402/682 (58.9%) in late 2020 (p < .001). Crude 28-day mortality decreased between the early and late periods (112/571 [19.6%] vs. 55/402 [13.7%]) and remained lower in the late period even after adjusting for baseline comorbidities (aOR 0.67, 95% CI 0.46-0.98, p = .016). Between the early and late periods, the use of corticosteroids (≥6 mg dexamethasone/day) and remdesivir increased (62/571 [10.9%] vs. 243/402 [61.5%], p < .001 and 50/571 [8.8%] vs. 213/402 [52.2%], p < .001, respectively), and the use of hydroxychloroquine and IL-6/IL-6 receptor inhibitor decreased (329/571 [60.0%] vs. 4/492 [1.0%], p < .001 and 73/571 [12.8%] vs. 5/402 [1.2%], p < .001, respectively). Mortality among SOTR hospitalized for COVID-19 declined between early and late 2020, consistent with trends reported in the general population. The mechanism(s) underlying improved survival require further study.

COVID-19 in hospitalized lung and non-lung solid organ transplant recipients: A comparative analysis from a multicenter study.

Lung transplant recipients (LTR) with coronavirus disease 2019 (COVID-19) may have higher mortality than non-lung solid organ transplant recipients (SOTR), but direct comparisons are limited. Risk factors for mortality specifically in LTR have not been explored. We performed a multicenter cohort study of adult SOTR with COVID-19 to compare mortality by 28 days between hospitalized LTR and non-lung SOTR. Multivariable logistic regression models were used to assess comorbidity-adjusted mortality among LTR vs. non-lung SOTR and to determine risk factors for death in LTR. Of 1,616 SOTR with COVID-19, 1,081 (66%) were hospitalized including 120/159 (75%) LTR and 961/1457 (66%) non-lung SOTR (p = .02). Mortality was higher among LTR compared to non-lung SOTR (24% vs. 16%, respectively, p = .032), and lung transplant was independently associated with death after adjusting for age and comorbidities (aOR 1.7, 95% CI 1.0-2.6, p = .04). Among LTR, chronic lung allograft dysfunction (aOR 3.3, 95% CI 1.0-11.3, p = .05) was the only independent risk factor for mortality and age >65 years, heart failure and obesity were not independently associated with death. Among SOTR hospitalized for COVID-19, LTR had higher mortality than non-lung SOTR. In LTR, chronic allograft dysfunction was independently associated with mortality.

Partial-AZFc deletions in Chilean men with primary spermatogenic impairment: gene dosage and Y-chromosome haplogroups.

PURPOSE: To investigate the association of partial-AZFc deletions in Chilean men with primary spermatogenic failure and their testicular histopathological phenotypes, analyzing the contribution of DAZ dosage, CDY1 copies, and Y-chromosome haplogroups. SUBJECTS AND METHODS: We studied 479 Chilean men: 334 infertile patients with histological examination (233 cases with spermatogenic defects and 101 normal spermatogenesis, obstructive controls, OC), and 145 normozoospermic controls (NC). AZFc subdeletions were detected by single-tagged sequences and single nucleotide variants analysis. DAZ-copy number was quantified by real-time qPCR. Y-chromosome haplogroups (Y-hg) were hierarchically genotyped through 16 biallelic-markers. RESULTS: The prevalence of AZFc-partial deletions was increased in cases (6%) compared with NC (1.4%) (P = 0.035). There was no difference between 143 Sertoli-cell only syndrome, 35 maturation arrest, or 35 mix atrophy patients and controls. However, gr/gr deletions were more frequent in 16 subjects with hypospermatogenesis compared with NC (P = 0.003) and OC (P = 0.013). Y-hg R was the most prevalent (~ 50%), but decreased among gr/gr deletions (21%, P = 0.03). The prevalence of Y-hg M increased in cases versus controls, both in total and non-deleted men (3.9 and 3.7% versus 0.4%, P = 0.009 and P = 0.016, respectively). Among gr/gr deletions, Y-hg H increased compared with non-deleted men (14.3% versus 0.4%, P = 0.0047). CONCLUSION: Partial-AZFc deletions in a Chilean admixed population are associated with secretory azo/oligozoospermia and might have a role in the development of hypospermatogenesis. Low represented haplogroups, Y-hg M and Y-hg H, show an association with the occurrence of spermatogenic failure and gr/gr deletions respectively; however, additional studies are required.

An Online Geographic Data Visualization Tool to Relate Preterm Births to Environmental Factors.

Preterm birth (<37 weeks gestation) continues to be a significant cause of disease and death in the United States. Its complex causes are associated with several genetic, biological, environmental, and sociodemographic factors. Organizing and visualizing various data that may be related to preterm birth is an essential step for pattern exploration and hypothesis generation and presents an opportunity to increase public and stakeholder involvement. In this article, we describe a collaborative effort to create an online geographic data visualization tool using open software to explore preterm birth in Fresno County, where rates are the highest in California. The tool incorporates information on births, environmental exposures, sociodemographic characteristics, the built environment, and access to care. We describe data sets used to build the tool, the data-hosting platform, and the process used to engage stakeholders in its creation. We highlight an important example of how collaboration can increase the utility of geographic data visualization to improve public health and address health equity in birth outcomes.

The need for transparency and good practices in the qPCR literature.

Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not.

Lower tacrolimus trough levels are associated with subsequently higher acute rejection risk during the first 12 months after kidney transplantation.

The premise that lower TAC trough levels are associated with subsequently higher first BPAR risk during the first 12 mo post-transplant was recently questioned. Using our prospectively followed cohort of 528 adult, primary kidney transplant recipients (pooled across four randomized trials) who received reduced TAC dosing plus an IMPDH inhibitor, TAC trough levels measured at seven time points, 7, 14 days, 1, 2, 3, 6 and 9 months post-transplant, were utilized along with Cox's model to determine the multivariable significance of TAC level(t) (a continuous time-dependent covariate equaling the most recently measured TAC level prior to time t) on the hazard rate of developing first BPAR during the first 12 months post-transplant. The percentage developing BPAR during the first 12 months post-transplant was 10.2% (54/528). In univariable analysis, lower TAC level(t) was associated with a significantly higher BPAR rate (P = 0.00006), and its significance was maintained even after controlling for 2 significant baseline predictors (African-American/Hispanic Recipient and Developed DGF) in Cox's model (multivariable P = 0.0003). Use of a cutpoint, TAC level(t) <4.0 vs. ≥4.0 ng/ml, yielded an even greater association with BPAR rate (univariable and multivariable P < 0.000001), with an estimated hazard ratio of 6.33. These results suggest that TAC levels <4.0 ng/ml should be avoided during the first 12 months post-transplant when TAC is used in combination with fixed-dose mycophenolate with or without corticosteroids and induction therapy.

Single-centre study of 628 adult, primary kidney transplant recipients showing no unfavourable effect of new-onset diabetes after transplant.

AIMS/HYPOTHESIS: To better understand the implications of new-onset diabetes after transplant (NODAT), we used our prospectively followed cohort of 628 adult primary kidney transplant recipients to determine the prognostic impact of pretransplant diabetes and NODAT. METHODS: The study cohort consisted of all participants in four randomised immunosuppression trials performed at our centre since May 2000. For each cause-specific hazard analysed, Cox stepwise regression was used to determine a multivariable model of significant baseline predictors; the multivariable influence of having pretransplant diabetes and NODAT (t) (the latter defined as a zero-one, time-dependent covariate) was subsequently tested. Similar analyses of estimated glomerular filtration rate (eGFR) at 36 and 60 months post transplant were performed using stepwise linear regression. Finally, a repeated measures analysis of mean HbA1c as a function of diabetes category (pretransplant diabetes vs NODAT) and randomised trial (first to fourth) was performed. RESULTS: Median follow-up was 56 months post transplant. Patients with pretransplant diabetes comprised 23.4% (147/628), and 22.5% (108/481) of the remaining patients developed NODAT. Pretransplant diabetes had no prognostic influence on first biopsy-proven acute rejection and death-censored graft failure hazard rates, nor on eGFR, but was associated with significantly higher rates of death with a functioning graft (DWFG) (p = 0.003), DWFG due to a cardiovascular event (p = 0.005) and infection that required hospitalisation (p = 0.03). NODAT (t) had no unfavourable impact on any of these hazard rates nor on eGFR, with actuarial freedom from DWFG remaining at over 90% among patients in pre- and post-NODAT states at 72 months post transplant/NODAT. Mean HbA1c for patients in the first to fourth randomised trials, averaged across diabetes category, decreased by trial (7.28%, 6.92%, 6.87% and 6.64% [56.1, 52.1, 51.6 and 49.1 mmol/mol], respectively; p = 0.02). CONCLUSIONS/INTERPRETATION: Less-than-expected post-NODAT risk for graft loss and death may exist in the current climate of tighter glucose monitoring post transplant.

Multivariable risk of developing new onset diabetes after transplant-results from a single-center study of 481 adult, primary kidney transplant recipients.

BACKGROUND: Understanding the relative contributions of baseline demographics and immunosuppressive therapy on NODAT risk may help in developing preventive strategies. METHODS: Using our prospectively followed cohort of 481 adult, primary kidney transplant recipients without pre-transplant diabetes, we determined the significant baseline predictors for the hazard rate of developing NODAT via Cox stepwise regression. The multivariable influence of first BPAR (defined as a time-dependent covariate) was also tested. RESULTS: Median follow-up was 57 mo post-transplant; the overall percentage who developed NODAT was 22.5% (108/481). Four baseline predictors of a greater NODAT hazard rate were found (by order of selection): higher BMI (p < 0.000001), planned maintenance with SRL (p = 0.0003), non-white recipient (p = 0.0004), and older recipient age (p = 0.0004). Approximately one-half of the 106 patients in the highest demographic risk category (BMI ≥25 kg/m(2) , non-white race, and age at transplant ≥40 yr) developed NODAT; actuarial NODAT risk ranged from 10% to 30% in the lower demographic risk categories. First BPAR was also associated with significantly higher NODAT in multivariable analysis (p = 0.02)-the highly elevated NODAT rate observed during the first few months post-transplant and following first BPAR appears to demonstrate the diabetogenic effect of using high-dose (intravenous) corticosteroids. CONCLUSIONS: The disturbingly high NODAT rate found among patients having multiple demographic risk factors is still an important problem that awaits a better solution.

[Effectiveness of supervised breast self-examination as educational strategy]. / Efectividad de la autoexploración mamaria supervisada como estrategia educative.

BACKGROUND: Breast cancer is the leading cause of death from cancer in the female population; consequently, there are multiple prevention campaigns. Within these campaigns, a special emphasis is given on promoting monthly breast self-examination; however, many women have never received formal education on proper method of self-examination. OBJECTIVE: To establish if the educational intervention we propose improves the breast self-examination technique. MATERIAL AND METHODS: A descriptive longitudinal study that included 52 women aged 20-40 years, attending a Family Medicine Unit of the Mexican Institute of Social Security, who were evaluated about self-examination technique before and after educational intervention, measured on a scale of 0 to 16. Statistical analysis was made with descriptive statistics and Student's t test. RESULTS: The mean age was 30.76 ± 5.87 years. The mean baseline score was 3.13 ± 2.55. The final average score after a month of the educational intervention was 10.69 ± 2.74, which represents an increase in average score of 7.55 ± 3.53. There was a significant increase in assessment scores after the educational intervention (p < 0.001). CONCLUSIONS: "Supervised breast self-examination" technique showed an increase in the ability of self-examination in patients. It can be considered an effective complementary method of teaching breast self-examination.

Regeneration of the caudal fin of the evolutionary ancient tropical gar Atractosteus tropicus.

BACKGROUND: The tropical gar (Atractosteus tropicus), a member of the Lepisosteidae family, is native to regions extending from southeastern Mexico to southern Costa Rica. This species serves as a unique bridge between tetrapods and teleosts due to its phylogenetic position, slow evolutionary rate, dense genetic map, gene similarities with humans, and ease of laboratory cultivation. As a taxonomic sister group to teleosts like the zebrafish (Danio rerio), known for its high regenerative capacity, it remains unclear whether the tropical gar shares a similar ability for regeneration. RESULTS: This study aims to elucidate the caudal fin regeneration process in tropical gar through skeletal and histological staining methods. Juvenile specimens were observed over a two-month period, during which they were fed brine shrimp, and anesthetized with 1% eugenol for caudal fin amputation. Samples were collected at various days post-amputation (dpa). Alcian blue and alizarin red staining were used to highlight skeletal regeneration, particularly the formation of new cartilage, while histological staining with hematoxylin and eosin was performed to observe tissue regeneration at the amputation site. CONCLUSIONS: The findings reveal a remarkable ability for caudal fin regeneration in juvenile tropical gar. Given the Garfish evolutionary relationship with teleosts, this opens new avenues for research into tissue regeneration across different groups of Actinopterygii.

Development of a Randomized Trial Comparing ICP-Monitor-Based Management of Severe Pediatric Traumatic Brain Injury to Management Based on Imaging and Clinical Examination Without ICP Monitoring-Study Protocol.

BACKGROUND AND OBJECTIVES: Traumatic brain injury (TBI) is a major global public health problem. It is a leading cause of death and disability in children and adolescents worldwide. Although increased intracranial pressure (ICP) is common and associated with death and poor outcome after pediatric TBI, the efficacy of current ICP-based management remains controversial. We intend to provide Class I evidence testing the efficacy of a protocol based on current ICP monitor-based management vs care based on imaging and clinical examination without ICP monitoring in pediatric severe TBI. METHODS: A phase III, multicenter, parallel-group, randomized superiority trial performed in intensive care units in Central and South America to determine the impact on 6-month outcome of children aged 1-12 years with severe TBI (age-appropriate Glasgow Coma Scale score ≤8) randomized to ICP-based or non-ICP-based management. EXPECTED OUTCOMES: Primary outcome is 6-month Pediatric Quality of Life. Secondary outcomes are 3-month Pediatric Quality of Life, mortality, 3-month and 6-month Pediatric extended Glasgow Outcome Score, intensive care unit length of stay, and number of interventions focused on treating measured or suspected intracranial hypertension. DISCUSSION: This is not a study of the value of knowing the ICP in sTBI. This research question is protocol-based. We are investigating the added value of protocolized ICP management to treatment based on imaging and clinical examination in the global population of severe pediatric TBI. Demonstrating efficacy should standardize ICP monitoring in severe pediatric TBI. Alternate results should prompt reassessment of how and in which patients ICP data should be applied in neurotrauma care.

Development of a Randomized Trial Comparing ICP-Monitor-Based Management of Severe Pediatric Traumatic Brain Injury to Management Based on Imaging and Clinical Examination Without ICP Monitoring-Research Algorithms.

BACKGROUND AND OBJECTIVES: The efficacy of our current approach to incorporating intracranial pressure (ICP) data into pediatric severe traumatic brain injury (sTBI) management is incompletely understood, lacking data from multicenter, prospective, randomized studies. The National Institutes of Health-supported Benchmark Evidence from Latin America-Treatment of Raised Intracranial Pressure-Pediatrics trial will compare outcomes from pediatric sTBI of a management protocol based on ICP monitoring vs 1 based on imaging and clinical examination without monitoring. Because no applicable comprehensive management algorithms for either cohort are available, it was necessary to develop them. METHODS: A consensus conference involving the 21 intensivists and neurosurgeons from the 8 trial sites used Delphi-based methodology to formulate management algorithms for both study cohorts. We included recommendations from the latest Brain Trauma Foundation pediatric sTBI guidelines and the consensus-based adult algorithms (Seattle International Brain Injury Consensus Conference/Consensus Revised Imaging and Clinical Examination) wherever relevant. We used a consensus threshold of 80%. RESULTS: We developed comprehensive management algorithms for monitored and nonmonitored cohort children with sTBI. We defined suspected intracranial hypertension for the nonmonitored group, set minimum number and timing of computed tomography scans, specified minimal age-adjusted mean arterial pressure and cerebral perfusion pressure targets, defined clinical neuroworsening, described minimal requisites for intensive care unit management, produced tiered management algorithms for both groups, and listed treatments not routinely used. CONCLUSION: We will study these protocols in the Benchmark Evidence from Latin America-Treatment of Raised Intracranial Pressure-Pediatrics trial in low- and middle-income countries. Second, we present them here for consideration as prototype pediatric sTBI management algorithms in the absence of published alternatives, acknowledging their limited evidentiary status. Therefore, herein, we describe our study design only, not recommended treatment protocols.

R-RAS2 overexpression in tumors of the human central nervous system.

Malignant tumors of the central nervous system (CNS) are the 10th most frequent cause of cancer mortality. Despite the strong malignancy of some such tumors, oncogenic mutations are rarely found in classic members of the RAS family of small GTPases. This raises the question as to whether other RAS family members may be affected in CNS tumors, excessively activating RAS pathways. The RAS-related subfamily of GTPases is that which is most closely related to classical Ras and it currently contains 3 members: RRAS, RRAS2 and RRAS3. While R-RAS and R-RAS2 are expressed ubiquitously, R-RAS3 expression is restricted to the CNS. Significantly, both wild type and mutated RRAS2 (also known as TC21) are overexpressed in human carcinomas of the oral cavity, esophagus, stomach, skin and breast, as well as in lymphomas. Hence, we analyzed the expression of R-RAS2 mRNA and protein in a wide variety of human CNS tumors and we found the R-RAS2 protein to be overexpressed in all of the 90 CNS cancer samples studied, including glioblastomas, astrocytomas and oligodendrogliomas. However, R-Ras2 was more strongly expressed in low grade (World Health Organization grades I-II) rather than high grade (grades III-IV) tumors, suggesting that R-RAS2 is overexpressed in the early stages of malignancy. Indeed, R-RAS2 overexpression was evident in pre-malignant hyperplasias, both at the mRNA and protein levels. Nevertheless, such dramatic changes in expression were not evident for the other two subfamily members, which implies that RRAS2 is the main factor triggering neural transformation.

Enhancing adherence of Arcobacter butzleri after serial intraperitoneal passages in mice.

We investigated the possibility of enhancing the adherence capacity of four low-adherent Arcobacter butzleri strains after serial intraperitoneal passage (i.p.) in mice. All the strains enhanced their adherence capacity after the first passage, increasing their adhesion rates after each passage. These results suggest that i.p. passage enhances the expression of adherence in A. butzleri strains.

His-tag ß-galactosidase supramolecular performance.

ß-Galactosidase is an important biotechnological enzyme used in the dairy industry, pharmacology and in molecular biology. In our laboratory we have overexpressed a recombinant ß-galactosidase in Escherichia coli (E. coli). This enzyme differs from its native version (ß-GalWT) in that 6 histidine residues have been added to the carboxyl terminus in the primary sequence (ß-GalHis), which allows its purification by immobilized metal affinity chromatography (IMAC). In this work we compared the functionality and structure of both proteins and evaluated their catalytic behavior on the kinetics of lactose hydrolysis. We observed a significant reduction in the enzymatic activity of ß-GalHis with respect to ß-GalWT. Although, both enzymes showed a similar catalytic profile as a function of temperature, ß-GalHis presented a higher resistance to the thermal inactivation compared to ß-GalWT. At room temperature, ß-GalHis showed a fluorescence spectrum compatible with a partially unstructured protein, however, it exhibited a lower tendency to the thermal-induced unfolding with respect to ß-GalWT. The distinctively supramolecular arranges of the proteins would explain the effect of the presence of His-tag on the enzymatic activity and thermal stability.

[A clinical guideline for diagnosis and treatment of hydrocele in childhood]. / Guía de práctica clínica. Diagnóstico y tratamiento del hidrocele en los niños.

OBJECTIVE: To develop a guideline available to the medical staff of the first and second level of care, which includes recommendations based on the best available evidence about diagnosis and management of hydrocele in the pediatric patient. METHODS: Clinical questions were formulated and structured. Standardized sequence was established to search for practice guidelines from the clinical questions raised on diagnosis and treatment of hydrocele in children. The working group searched clinical practice guidelines and found only one. For recommendations not included in the reference guide the search process was conducted in PubMed and Cochrane Library. The results were expressed as levels of evidence and grade of recommendation. CONCLUSIONS: The most of the infant hydroceles are communicating and do resolve without treatment, however recognize those to need surgery is necessary to treat opportunely.

Biofabrication of functional protein nanoparticles through simple His-tag engineering.

We have developed a simple, robust, and fully transversal approach for the a-la-carte fabrication of functional multimeric nanoparticles with potential biomedical applications, validated here by a set of diverse and unrelated polypeptides. The proposed concept is based on the controlled coordination between Zn2+ ions and His residues in His-tagged proteins. This approach results in a spontaneous and reproducible protein assembly as nanoscale oligomers that keep the original functionalities of the protein building blocks. The assembly of these materials is not linked to particular polypeptide features, and it is based on an environmentally friendly and sustainable approach. The resulting nanoparticles, with dimensions ranging between 10 and 15 nm, are regular in size, are architecturally stable, are fully functional, and serve as intermediates in a more complex assembly process, resulting in the formation of microscale protein materials. Since most of the recombinant proteins produced by biochemical and biotechnological industries and intended for biomedical research are His-tagged, the green biofabrication procedure proposed here can be straightforwardly applied to a huge spectrum of protein species for their conversion into their respective nanostructured formats.

Aequorin functional assay for characterization of G-protein-coupled receptors: implementation with cryopreserved transiently transfected cells.

Assay technologies that measure intracellular Ca(2+) release are among the predominant methods for evaluation of GPCR function. These measurements have historically been performed using cell-permeable fluorescent dyes, although the use of the recombinant photoprotein aequorin (AEQ) as a Ca(2+) sensor has gained popularity with recent advances in instrumentation. The requirement of the AEQ system for cells expressing both the photoprotein and the GPCR target of interest has necessitated the labor-intensive development of cell lines stably expressing both proteins. With the goal of streamlining this process, transient transfections were used to either (1) introduce AEQ into cells stably expressing the GPCR of interest or (2) introduce the GPCR into cells stably expressing the AEQ protein, employing the human muscarinic M(1) receptor as a model system. Robust results were obtained from cryopreserved cells prepared by both strategies, yielding agonist and antagonist pharmacology in good agreement with literature values. Good reproducibility was observed between multiple transient transfection events. These results indicate that transient transfection is a viable and efficient method for production of cellular reagents for use in AEQ assays.

Superactive ß-galactosidase inclusion bodies.

Bacterial inclusion bodies (IBs) were historically considered one of the major obstacles in protein production through recombinant DNA techniques and conceived as amorphous deposits formed by passive and rather unspecific structures of unfolded proteins aggregates. Subsequent studies demonstrated that IBs contained an important quantity of active protein. In this work, we proved that recombinant ß-galactosidase inclusion bodies (IBß-Gal) are functional aggregates. Moreover, they exhibit particular features distinct to the soluble version of the enzyme. The particulate enzyme was highly active against lactose in physiological and in acid pH and also retained its activity upon a pre-incubation at high temperature. IBß-Gal washing or dilution induced the spontaneous release of active enzymes from the supramolecular aggregates. Along this process, we observed a continuous change in the values of several kinetic parameters, including specific activity and Michaelis-Menten constant, measured in the IBß-Gal suspensions. Simultaneously, IBß-Gal turned into a more heterogeneous population where smaller particles appeared. The released protein exhibited secondary structure features more similar to those of the soluble species than to the aggregated enzyme. Concluding, IBß-Gal represents a reservoir and packed source of highly active and stable enzyme.