Hydromorphone impurity 2,2-bishydromorphone does not exert mutagenic and clastogenic properties via in silico QSAR prediction and in vitro Ames and micronucleus test.

The opioid agonist hydromorphone is indicated for the management of severe acute and chronic pain given that alternate treatments are insufficient. While the genotoxicity profile of hydromorphone is well investigated, little is known about the genotoxic potential of its impurities. In this study, 2,2-bishydromorphone was tested in silico and in vitro for both its mutagenic potential in an Ames test performed with Salmonella typhimurium and Escherichia coli tester strains up to a maximum concentration of 5 mg per plate in the absence and presence of metabolic activation. Furthermore, it was tested for its ability to induce micronuclei in TK6 cells in a micronucleus test up to a maximum concentration of 500 µg/mL with or without an exogenous metabolic activation system. 2,2-Bishydromorphone did not reveal any potential for inducing mutagenicity or clastogenicity under the conditions of the respective tests and is therefore considered non-mutagenic and non-clastogenic/aneugenic in vitro. These results are in line with negative in silico quantitative structure-activity relationship (QSAR) prediction for 2,2-bishydromorphone mutagenicity and clastogenicity and provide evidence of good correlation of in silico and in vitro data. Conclusively, these studies add important new clinically relevant information on the safety of hydromorphone as the impurity of 2,2-bishydromorphone is proven to be non-mutagenic and non-clastogenic.

Key Challenges and Recommendations for In Vitro Testing of Tobacco Products for Regulatory Applications: Consideration of Test Materials and Exposure Parameters.

The Institute for In Vitro Sciences (IIVS) is sponsoring a series of workshops to identify, discuss and develop recommendations for optimal scientific and technical approaches for conducting in vitro assays, to assess potential toxicity within and across tobacco and various next generation nicotine and tobacco products (NGPs), including heated tobacco products (HTPs) and electronic nicotine delivery systems (ENDS). The third workshop (24-26 February 2020) summarised the key challenges and made recommendations concerning appropriate methods of test article generation and cell exposure from combustible cigarettes, HTPs and ENDS. Expert speakers provided their research, perspectives and recommendations for the three basic types of tobacco-related test articles: i) pad-collected material (PCM); ii) gas vapour phase (GVP); and iii) whole smoke/aerosol. These three types of samples can be tested individually, or the PCM and GVP can be combined. Whole smoke/aerosol can be bubbled through media or applied directly to cells at the air-liquid interface. Summaries of the speaker presentations and the recommendations developed by the workgroup are presented. Following discussion, the workshop concluded the following: that there needs to be greater standardisation in aerosol generation and collection processes; that methods for testing the NGPs need to be developed and/or optimised, since simply mirroring cigarette smoke testing approaches may be insufficient; that understanding and quantitating the applied dose is fundamental to the interpretation of data and conclusions from each study; and that whole smoke/aerosol approaches must be contextualised with regard to key information, including appropriate experimental controls, environmental conditioning, analytical monitoring, verification and performance criteria.

Negative immune responses to two-dose mRNA COVID-19 vaccines in renal allograft recipients assessed with simple antibody and interferon gamma release assay cellular monitoring.

Studies are urgently needed to characterize immunogenicity, efficacy, and safety of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccines in kidney transplant (KT) recipients, excluded from major clinical trials. Complex ELISPOT and other cellular response techniques have been applied, but simpler tools are needed. An easy-to-use real-world monitoring of SARS-CoV-2 IgG antibodies against the Spike protein and QuantiFERON® SARS-CoV-2 IFNγ release assay (IGRA) were performed at baseline and 28 days after the second dose in KT recipients and controls (dialysis patients and healthy ones). All healthy controls and >95% dialysis controls became positive for anti-S IgG antibodies, while only 63.3% of KT patients seroconverted with a very low antibody level. A positive IGRA was documented in 96.9% of controls, 89.3% peritoneal dialysis, 77.6% hemodialysis, 61.3% of KT patients transplanted more than 1 year ago and only 36% of those transplanted within the previous 12 months. Overall, 100% of healthy controls, 95.4% of dialysis patients and 78.8% KT recipients developed any immune response (humoral and/or cellular) against SARS-CoV-2. KT patients showed low rates of immune responses to mRNA Coronavirus infectious disease 2019 vaccines, especially those with recent transplantations. Simple humoral and cellular monitoring is advisable, so that repeated doses may be scheduled according to the results.

Meiosis-induced alterations in transcript architecture and noncoding RNA expression in S. cerevisiae.

Changes in transcript architecture can have powerful effects on protein expression. Regulation of the transcriptome is often dramatically revealed during dynamic conditions such as development. To examine changes in transcript architecture we analyzed the expression and transcript boundaries of protein-coding and noncoding RNAs over the developmental process of meiosis in Saccharomyces cerevisiae. Custom-designed, high-resolution tiling arrays were used to define the time-resolved transcriptome of cells undergoing meiosis and sporulation. These arrays were specifically designed for the S. cerevisiae strain SK1 that sporulates with high efficiency and synchrony. In addition, new methods were created to define transcript boundaries and to identify dynamic changes in transcript expression and architecture over time. Of 8407 total segments, 699 (8.3%) were identified by our algorithm as regions containing potential transcript architecture changes. Our analyses reveal extensive changes to both the coding and noncoding transcriptome, including altered 5' ends, 3' ends, and splice sites. Additionally, 3910 (46.5%) unannotated expressed segments were identified. Interestingly, subsets of unannotated RNAs are located across from introns (anti-introns) or across from the junction between two genes (anti-intergenic junctions). Many of these unannotated RNAs are abundant and exhibit sporulation-specific changes in expression patterns. All work, including heat maps of the tiling array, annotation for the SK1 strain, and phastCONS conservation analysis, is available at http://groups.molbiosci.northwestern.edu/sontheimer/sk1meiosis.php. Our high-resolution transcriptome analyses reveal that coding and noncoding transcript architectures are exceptionally dynamic in S. cerevisiae and suggest a vast array of novel transcriptional and post-transcriptional control mechanisms that are activated upon meiosis and sporulation.

Dynamics of Humoral and Cellular Responses in Renal Transplant Recipients Receiving 3 Doses of SARS-CoV-2 mRNA Vaccine.

BACKGROUND: The original SARS-CoV-2 vaccination regimen (2 doses) induces insufficient short-term response in kidney transplant (KT) recipients. This study assessed the response to a third dose and the long-term immunogenicity after 2 doses in KT. METHODS: We analyzed the dynamics of the humoral and cellular response by monitoring SARS-CoV-2 IgG antibodies against the Spike-protein (IgG-Spike) and QuantiFERON SARS-CoV-2 IFN-γ release assay 6 mo after the second dose (T2) and 28 d after the third dose of mRNA vaccines (T3) to KT and controls (dialysis patients and healthy individuals). RESULTS: At T2, the percentage of IgG-Spike+ KT and dialysis patients decreased (KT 65.8%-52.6%, hemodialysis 92.6-81.5%, and peritoneal dialysis 100%-90%), whereas 100% of healthy controls remained positive. About the cellular response, the percentage of responders decreased in all groups, especially in KT (22.4%-9.2%, P = 0.081). At T3, 92% of KT, 94%-98% of dialysis patients, and 100% of healthy controls were IgG-Spike+. In terms of antibody titers, patients and controls showed a reduction between T2 and T3 and about 80% of dialysis patients and 100% of controls achieved high titers after the third dose (>1479.5 Binding Antibody Units/mL), whereas this percentage was only 50% in KT. With respect to the cellular response, only KT displayed a significant rise after the third dose. CONCLUSIONS: The third dose of mRNA vaccine improves both humoral and cellular responses, but less effectively in KT compared with dialysis patients and healthy controls.

Proteomics identification of Drosophila small interfering RNA-associated factors.

The Drosophila melanogaster RNA-induced silencing complex (RISC) forms a large ribonucleoprotein particle on small interfering RNAs (siRNAs) and catalyzes target mRNA cleavage during RNA interference (RNAi). Dicer-2, R2D2, Loquacious, and Argonaute-2 are examples of RISC-associated factors that are involved in RNAi. Holo-RISC is an approximately 80 S small interfering ribonucleoprotein, which suggests that there are many additional proteins that participate in the RNAi pathway. In this study, we used siRNA affinity capture combined with mass spectrometry to identify novel components of the Drosophila RNAi machinery. Our study identified both established RISC components and novel siRNA-associated factors, many of which contain domains that are consistent with potential roles in RNAi. Functional analysis of these novel siRNA-associated proteins suggests that these factors may play an important role in RNAi.

Three regulatory compliant test systems show no signs of MDMA-related genotoxicity.

3,4 Methylenedioxymethamphetamine (MDMA)-assisted therapy has been recently found to be highly effective for treatment of posttraumatic stress disorder (PTSD). Previous studies have been inconclusive in elucidating potential MDMA genotoxicity. We performed three regulatory compliant studies to investigate the potential of genotoxic effects of MDMA treatment in humans: (1) an in vitro bacterial reverse mutation (Ames) assay, (2) an in vitro chromosome aberration test in Chinese hamster ovary cells, and (3) an in vivo micronucleus study in male Sprague Dawley rats. MDMA was found to not have genotoxic effects in any of the assays at or above clinically relevant concentrations.

Mortality in Hemodialysis Patients with COVID-19, the Effect of Paricalcitol or Calcimimetics.

BACKGROUND: In COVID-19 patients, low serum vitamin D (VD) levels have been associated with severe acute respiratory failure and poor prognosis. In regular hemodialysis (HD) patients, there is VD deficiency and markedly reduced calcitriol levels, which may predispose them to worse outcomes of COVID-19 infection. Some hemodialysis patients receive treatment with drugs for secondary hyperparathyroidism, which have well known pleiotropic effects beyond mineral metabolism. The aim of this study was to evaluate the impact of VD status and the administration of active vitamin D medications, used to treat secondary hyperparathyroidism, on survival in a cohort of COVID-19 positive HD patients. METHODS: A cross-sectional retrospective observational study was conducted from 12 March to 21 May 2020 in 288 HD patients with positive PCR for SARS-CoV2. Patients were from 52 different centers in Spain. RESULTS: The percent of HD patients with COVID-19 was 6.1% (288 out of 4743). Mortality rate was 28.4% (81/285). Three patients were lost to follow-up. Serum 25(OH)D (calcidiol) level was 17.1 [10.6-27.5] ng/mL and was not significantly associated to mortality (OR 0.99 (0.97-1.01), p = 0.4). Patients receiving active vitamin D medications (16/94 (17%) vs. 65/191(34%), p = 0.003), including calcimimetics (4/49 (8.2%) vs. 77/236 (32.6%), p = 0.001), paricalcitol or calcimimetics (19/117 (16.2%) vs. 62/168 (36.9%); p < 0.001), and also those on both paricalcitol and calcimimetics, to treat secondary hyperparathyroidism (SHPTH) (1/26 (3.8%) vs. 80/259 (30.9%), p < 0.001) showed a lower mortality rate than patients receiving no treatment with either drug. Multivariate Cox regression analysis confirmed this increased survival. CONCLUSIONS: Our findings suggest that the use of paricalcitol, calcimimetics or the combination of both, seem to be associated with the improvement of survival in HD patients with COVID-19. No correlation was found between serum VD levels and prognosis or outcomes in HD patients with COVID-19. Prospective studies and clinical trials are needed to support these findings.

[Outcomes the Mitrofanoff technique in the management of patients with neurogenic bladder: the experience in the San Vicente de Paul Universitary Hospital]. / Resultados del uso de la técnica de Mitrofanoff en pacientes con disfunción neurógena de la vejiga: análisis de la experiencia en el Hospital Universitario San Vicente de Paul.

INTRODUCTION: The care for children with neurogenic bladder, should be integral, multidisciplinary look to preserve renal function and to accomplish urinary and fecal continence, achieving that the patient becomes self-sufficient and useful to society. METHODS: The result of the use of the Mitrofanoff technique for the treatment of patients with neurogenic dysfunction bladder used in the HUSVP 1998-2003, and the current condition of the patients with respect to their illness and treatment are described in this article. A descriptive retrospective study 1998-2002 and a prospective study during 2003 were done, in which a series of cases were analyzed. RESULTS: 41 patients had surgery. Average age 10.2 years; average follow-up time 27.2 months; the most frequent illness was myelomeningocele (46.3%) and 46.3% had dysinergic bladder. Bladder augmentation was performed on 63.3%, of which 71.4% were constructed with ileum. A surgical intervention of the bladder neck was done on 51.2%. A continent conduct (Mitrofanoff) was performed on 95.1% of the patients with complete continence 70.4%, complete incontinence 14.6% and occasional incontinence 14.6%. 31.7% had Malone surgery with adequate fecal management on 90.2%. 19% of the stomas presented stenosis and 21.9% presented urine leakage. 80% reached appropriate social adaptation. CONCLUSIONS: The continent catheterizable stomas are useful for the treatment of urinary and fecal incontinence. The conducts constructed with ileum had more complications than the conducts done with cecal appendix, which is why the appendix is the choice tissue to perform the continent catheterizable stomas, as long as it is available.

SPO24 is a transcriptionally dynamic, small ORF-encoding locus required for efficient sporulation in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, meiosis and sporulation are highly regulated responses that are driven in part by changes in RNA expression. Alternative mRNA forms with extended 5' UTRs are atypical in S. cerevisiae, and 5' extensions with upstream open reading frames (uORFs) are even more unusual. Here we characterize the gene YPR036W-A, now renamed SPO24, which encodes a very small (67-amino-acid) protein. This gene gives rise to two mRNA forms: a shorter form throughout meiosis and a longer, 5'-extended form in mid-late meiosis. The latter form includes a uORF for a 14-amino-acid peptide (Spo24u14). Deletion of the downstream ORF (dORF) leads to sporulation defects and the appearance of pseudohyphae-like projections. Experiments with luciferase reporters indicate that the uORF does not downregulate dORF translation. The protein encoded by the dORF (Spo24d67) localizes to the prospore membrane and is differentially phosphorylated during meiosis. Transcription of the 5'-extended mRNA in mid-meiosis depends upon the presence of two middle sporulation elements (MSEs). Removal of the MSEs severely inhibits the mid-meiotic appearance of the 5'-extended mRNA and limits the ability of plasmid-borne SPO24 to rescue the sporulation defect of a spo24Δ mutant, suggesting that the 5'-extended mRNA is functionally important. These results reveal Spo24d67 as a sporulation-related factor that is encoded by a transcriptionally dynamic, uORF-containing locus.

Resultados del uso de la técnica de Mitrofanoff en pacientes con disfunción neurógena de la vejiga: Análisis de la experiencia en el Hospital Universitario San Vicente de Paul / Outcomes the Mitrofanoff technique in the management of patients with neurogenic bladder: the experience in the San Vicente de Paul Universitary Hospital

Introducción: El cuidado de los niños con vejiga neurogénica, debe ser integral, multidisciplinario y busca preservar la función renal, alcanzar continencia urinaria y fecal, logrando que el paciente sea una persona autosuficiente y útil a la sociedad. Métodos: Describimos el resultado del uso de la técnica de Mitrofanoff en el manejo de pacientes con disfunción vesical neurógena, utilizada en el HUSVP desde 1998 al 2003, y las condiciones actuales de los pacientes con respecto a su enfermedad y tratamiento. Se realizó un estudio observacional descriptivo, retrospectivo 1998-2002 y prospectivo durante el 2003, analizando una serie de casos. Resultados: Fueron intervenidos 41 pacientes con vejiga neurogénica. Edad promedio 10.2 años; seguimiento promedio 27,2 meses; enfermedad de base más frecuente mielomeningocele (46,3%); vejiga disinérgica en 46,3%, esfínter hipotónico en 41,5%. Se realizo aumento vesical al 68,3%, utilizándose ileon en 71,4%, y alguna intervención sobre el cuello vesical al 51,2%. Se hizo una derivación tipo Mitrofanoff al 95,1% con continencia completa en 70,8%, incontinencia completa en 14.6% e incontinencia ocasional en 14,6%. En 31,7% se realizó un Malone con adecuada continencia fecal en 90,2%. De los estomas el 19% presentaron estenosis y el 21,9% presentaron escape de orina. Se encuentran socialmente adaptados 80% de los pacientes. Conclusiones: Los estomas continentes cateterizables son útiles para tratar la incontinencia urinaria y fecal. Los conductos realizados con ileon tuvieron mayores complicaciones que los elaborados con apéndice, por lo cual el apéndice debe ser el tejido de elección para realizar las derivaciones, cuando esté disponible (AU)

Introduction: The care for children with neurogenic bladder, should be integral, multidisciplinary look to preserve renal function and to accomplish urinary and fecal continence, achieving that the patient becomes self-sufficient and useful to society. Methods: The result of the use of the Mitrofanoff technique for the treatment of patients with neurogenic dysfunction bladder used in the HUSVP 1998- 2003, and the current condition of the patients with respect to their illness and treatment are described in this article. A descriptive retrospective study 1998- 2002 and a prospective study during 2003 were done, in which a series of cases were analyzed. Results: 41 patients had surgery. Average age 10.2 years; average follow–up time 27.2 months; the most frequent illness was myelomeningocele (46.3%) and 46.3% had dysinergic bladder. Bladder augmentation was performed on 63.3%, of which 71.4% were constructed with ileum. A surgical intervention of the bladder neck was done on 51.2%. A continent conduct (Mitrofanoff) was performed on 95.1% of the patients with complete continence 70.4%, complete incontinence 14.6% and occasional incontinence 14.6%. 31.7% had Malone surgery with adequate fecal management on 90.2%. 19% of the stomas presented stenosis and 21.9% presented urine leakage. 80% reached appropriate social adaptation. Conclusions: The continent catheterizable stomas are useful for the treatment of urinary and fecal incontinence. The conducts constructed with ileum had more complications than the conducts done with cecal appendix, which is why the appendix is the choice tissue to perform the continent catheterizable stomas, as long as it is available (AU)