A dual-reference study design for understanding and improving AAV genome size analysis.

Recombinant adeno-associated virus (rAAV) is the leading platform of gene delivery for its long-lasting gene transformation and low immunogenicity. Characterization of the integrity and purity of the rAAV genome is critical to ensure clinical potency and safety. However, current rAAV genome characterization methods that can provide size assessment are either time-consuming or not easily accessible to general labs. Additionally, there is a lack of right reference standard for analyzing long single-stranded DNA (ssDNA) fragments. Here, we have developed an ssDNA assay on a microfluidic capillary electrophoresis platform using ssDNA reference standard. This assay provides size calling for ssDNA fragment, a detection sensitivity at ∼89 pg/µL (3 × 1010 GC/mL AAV) for 5.1 kb ssDNA fragment, and a turnaround time at ∼100 s per sample with a high throughput sample analyzing capability. Moreover, we have observed that the annealing of AAV ssDNA subsequent to its release from the capsid might introduce an additional double-stranded DNA (dsDNA) peak. This phenomenon is dependent on the sample processing workflow. To avoid the risk of mischaracterization, we recommend the use of dual-reference standards in combination with other orthogonal methods to have a comprehensive understanding of the rAAV genome size and integrity.

Purity and DNA content of AAV capsids assessed by analytical ultracentrifugation and orthogonal biophysical techniques.

Development and manufacturing adeno-associated virus (AAV)-based vectors for gene therapy requires suitable analytical methods to assess the quality of the formulations during development, as well as the quality of different batches and the consistency of the processes. Here, we compare biophysical methods to characterize purity and DNA content of viral capsids from five different serotypes (AAV2, AAV5, AAV6, AAV8, and AAV9). For this purpose, we apply multiwavelength sedimentation velocity analytical ultracentrifugation (SV-AUC) to obtain the species' contents and to derive the wavelength-specific correction factors for the respective insert-size. In an orthogonal manner we perform anion exchange chromatography (AEX) and UV-spectroscopy and the three methods yield comparable results on empty/filled capsid contents with these correction factors. Whereas AEX and UV-spectroscopy can quantify empty and filled AAVs, only SV-AUC could identify the low amounts of partially filled capsids present in the samples used in this study. Finally, we employ negative-staining transmission electron microscopy and mass photometry to support the empty/filled ratios with methods that classify individual capsids. The obtained ratios are consistent throughout the orthogonal approaches as long as no other impurities and aggregates are present. Our results show that the combination of selected orthogonal methods can deliver consistent empty/filled contents on non-standard genome sizes, as well as information on other relevant critical quality attributes, such as AAV capsid concentration, genome concentration, insert size length and sample purity to characterize and compare AAV preparations.

Working Anytime and Anywhere -Even When Feeling Ill? A Cross-sectional Study on Presenteeism in Remote Work.

Background: Working despite feeling ill - presenteeism - is a widespread behavioral phenomenon. Previous research has shown that presenteeism is influenced by various work-related and personal factors. It's an illness behavior leading to a range of negative but also positive consequences. Due to coronavirus disease 2019 (COVID-19) pandemic, remote work has become the "new normal" for many employees. But so far, little is known about presenteeism in remote work. This study aims to investigate presenteeism in remote work by looking at the extent of remote presenteeism, differences to presenteeism in on-site work, and associated factors. Methods: A nationwide cross-sectional online survey was conducted in Germany with N = 233 participants. Data were analyzed using descriptive statistics, t-tests, and correlation analysis. Results: The results reveal that presenteeism is prevalent in remote work x̅ = 4.13 days (Md = 3; D = 2; s = 4.95). A low ability to detach from work (r = -.17; p = .005) and low supervisor support (r = -.14; p = .02) is associated with more remote presenteeism days. Remote working conditions seem to facilitate presenteeism. Conclusion: This study provides empirical insights into a subject area of great societal relevance. The results show that awareness should be raised for presenteeism in remote work. It should be regarded as a behavior that can be functional or dysfunctional, depending on the individual situation. Supervisor support and detachment should be fostered to help reduce dysfunctional presenteeism. Promotion of health literacy might help remote workers to decide on a health-oriented illness behavior. Further research is vital to analyze to what extent and under which circumstances presenteeism in remote work is (dys)functional and to derive clear recommendations.

Stable bone density in adolescents with severe growth hormone deficiency after six months off rhGH.

OBJECTIVE: Severe growth hormone deficiency causes lean body mass loss in male adolescents and increased fat mass in both sexes. The changes appear after a 6 month GH pause. AIM: The aim was to examine bone density and structure changes in adolescents with severe GHD during a 6-month rhGH treatment interruption. PATIENTS AND METHODS: In total, 113 adolescents (20 females) paused rhGH treatment for 6 months at near-final height, and they were retested with arginine-GHRH challenge and basal IGF-1. Severe GHD was diagnosed in 19 individuals (5 females, GH peak <16 ng/ml and IGF-1 < -1.9 SDS) and excluded in 94 (15 females). Bone density and structure were measured by pQCT of the forearm and DXA of the total body at cessation of rhGH and 6 months later. RESULTS: In severe adolescent GHD (sGHD) patients, trabecular density (mg/cm3) decreased from 214 to 202 (p < 0.01); changes in the adolescents with normal test results (tGHD) were from 221 to 214 (p < 0.05). Cortical density (mg/cm3) increased from 1077 to 1099 (p < 0.01) in sGHD patients and from 1060 to 1082 in tGHD patients (p < 0.001). The strength strain index (mm3) showed no significant changes in sGHD patients (306 to 307) but changed from 302 to 315 in tGHD patients (p < 0.05). Total bone area (mm2) shifted from 145.1 to 145.2 in sGHD patients and from 153 to 156 in tGHD patients. Total body aBMD (g/cm2) increased in both groups: from 1.10 to 1.12 in sGHD patients and from 1.11 to 1.14 in tGHD patients (p < 0.01). All bone measurements remained within the reference ranges, and there were no differences between sGHD and tGHD patients. CONCLUSION: During a 6-month pause of rhGH treatment, the bone structure and density of adolescents with sGHD did not show changes implying harm. Routine retesting of adolescents, including 6 months without GH, is unlikely to be detrimental to the bone.

Changes in body composition in male adolescents with childhood-onset GH deficiency during transition.

OBJECTIVE: Restarting rhGH in adolescents with childhood-onset (CO-) GHD is usually based on GH retest, IGF-1, additional pituitary hormone deficiencies, pituitary morphology and history. Short-term changes in body composition in adolescents with CO-GHD when off rhGH may contribute to the identification of those in need of treatment continuation. DESIGN: This is a longitudinal single-centre study. PATIENTS AND MEASUREMENTS: The body composition of 90 male adolescents with low-likelihood severe GHD of adolescence was measured by DXA at the time of rhGH discontinuation and 6 months thereafter. At diagnosis, mean age was 5.4 years, height was -2.68 SDS and stimulated GH peak was 5.1 ng/mL. RhGH treatment was stopped at 16.7 years at near-final height of -0.44 SDS. The adolescents were re-examined after 3 months off rhGH using both IGF-1 and GHRH-arginine tests. Severe GHD of adolescence was defined both by stimulated GH < 16 ng/mL and by IGF-1 < -1.90 SDS. RESULTS: Males with severe GHD of adolescence (n = 8) gained more relative and absolute fat mass and lost significantly more relative lean body mass after 6 months off rhGH than healthy individuals (n = 82; P < 0.001). The sum of absolute fat mass gain and lean body mass loss (=body composition changes score; BCC score) correlated highly with the GH peak (R = 0.17; P < 0.001). A BCC score >7.0 kg was 88% sensitive and 94% specific for detecting severe GHD of adolescence (AUC = 0.975). CONCLUSIONS: Short-term body composition changes when off rhGH are good clinical markers of severe GHD in male adolescents. The novel BBC score is an aggregate of these changes.

Success of kidney transplantations from deceased donors with acute kidney injury.

BACKGROUND The acceptance of organs from deceased donors with acute kidney injury (AKI) varies considerably, with uncertain outcomes. The current organ shortage has led to increased use of marginal donor organs. MATERIAL AND METHODS This retrospective, single-center study included 642 patients who underwent kidney allograft transplantation between 2005 and 2016. The recipients were categorized into 3 groups: AKI-1 (n=214), comprising donors with a peak serum creatinine (SCr) level of 1.1-2.0 mg/dl; AKI-2 (n=89), comprising donors with a peak SCr level >2 mg/dl; and non-AKI (n=339), comprising donors with normal kidney function (SCr <1.1 mg/dl). RESULTS The cumulative survival rates for patients and grafts did not significantly differ among the AKI-1, AKI-2, and non-AKI groups at the 1-year (91.6%/79.4%, 92.1%/83.1%, 95.3%/88.5%, respectively) and 5-year assessments (79.4%/67.8%, 86.8%/71.7%, 80.5%/71.1%, respectively). These findings were corroborated by mean SCr values and estimated glomerular filtration rates at the 1-year (2.08±1.7/51.16±23.45, 2.01±1.52/56.46±23.63, 1.81±1.13/55.44±23.26 mg/dl, respectively) and 5-year assessments (1.91±1.28/51.06±24.65, 1.74±0.66/57.44±31.21, 1.7±0.88/58.56±26.04 mg/dl, respectively). The incidence of delayed graft function in each group was 29.9%, 44.9%, and 28.6%, respectively. CONCLUSIONS Kidney transplantation from donors with AKI, although associated with a higher rate of delayed graft function, results in good long-term transplant survival and reliable kidney functionality after 5 years. The inclusion of donors with AKI may widely extend the pool of available organs; however, careful donor selection is necessary.

A multitracer test proving the reliability of Rayleigh equation-based approach for assessing biodegradation in a BTEX contaminated aquifer.

Compound-specific stable isotope analysis (CSIA) is one of the most important methods for assessing biodegradation activities in contaminated aquifers. Although the concept is straightforward, the proof that the method cannot be only used for a qualitative analysis but also to quantify biodegradation in the subsurface was missing. We therefore performed a multitracer test in the field with ring-deuterated (d5) and completely (d8) deuterium-labeled toluene isotopologues (400 g) as reactive tracers as well as bromide as a conservative tracer. The compounds were injected into the anoxic zone of a BTEX plume located down-gradient of the contaminant source. Over a period of 4.5 months the tracer concentrations were analyzed at two control planes located 24 and 35 m downgradient of the injection well. Deuterium-labeled benzylsuccinate was found in the aquifer, indicating the anaerobic biodegradation of deuterated toluene via the benzylsuccinate synthase pathway. Three independent methods were applied to quantify biodegradation of deuterated toluene. First, fractionation of toluene-d8 and toluene-d5 using the Rayleigh equation and an appropriate laboratory-derived isotope fractionation factor was used for the calculation of the microbial decomposition of deuterated toluene isotopologues (CSIA-method). Second, the biodegradation was quantified by the changes of the concentrations of deuterated toluene relative to bromide. Both methods gave similar results, implying that the CSIA-method is a reliable tool to quantify biodegradation in contaminated aquifers. The results of both methods yielded a biodegradation of deuterated toluene isotopologues of approximately 23-29% for the first and 44-51% for the second control plane. Third, the mineralization of deuterated toluene isotopologues was verified by determination of the enrichment of deuterium in the groundwater. This method indicated that parts of deuterium were assimilated into the biomass of toluene degrading microorganisms.