Synthesis and Structural Optimization of ATG4B Inhibitors for the Attenuation of Autophagy in Glioblastoma.

Glioblastoma, a prevalent malignant CNS tumor, presents a therapeutic challenge because of resistance to standard treatments, including radiation therapy and temozolomide. Both modalities induce autophagy, thereby paradoxically promoting tumor survival. The cysteine protease ATG4B is implicated in this cellular process, which highlights the enzyme as a viable therapeutic target for glioblastoma. We have developed streamlined syntheses for ATG4B inhibitor NSC185058, its derivatives, and fluorogenic ATG4B substrate pim-FG-PABA-AMC. We leveraged these findings to rapidly identify novel compound MJO445, which demonstrates markedly greater potency biochemically and in cells.

Treatment recommendations for glycogen storage disease type IB- associated neutropenia and neutrophil dysfunction with empagliflozin: Consensus from an international workshop.

Glycogen storage disease type Ib (GSD Ib, biallelic variants in SLC37A4) is a rare disorder of glycogen metabolism complicated by neutropenia/neutrophil dysfunction. Since 2019, the SGLT2-inhibitor empagliflozin has provided a mechanism-based treatment option for the symptoms caused by neutropenia/neutrophil dysfunction (e.g. mucosal lesions, inflammatory bowel disease). Because of the rarity of GSD Ib, the published evidence on safety and efficacy of empagliflozin is still limited and does not allow to develop evidence-based guidelines. Here, an international group of experts provides 14 best practice consensus treatment recommendations based on expert practice and review of the published evidence. We recommend to start empagliflozin in all GSD Ib individuals with clinical or laboratory signs related to neutropenia/neutrophil dysfunction with a dose of 0.3-0.4 mg/kg/d given as a single dose in the morning. Treatment can be started in an outpatient setting. The dose should be adapted to the weight and in case of inadequate clinical treatment response or side effects. We strongly recommend to pause empagliflozin immediately in case of threatening dehydration and before planned longer surgeries. Discontinuation of G-CSF therapy should be attempted in all individuals. If available, 1,5-AG should be monitored. Individuals who have previously not tolerated starches should be encouraged to make a new attempt to introduce starch in their diet after initiation of empagliflozin treatment. We advise to monitor certain safety and efficacy parameters and recommend continuous, alternatively frequent glucose measurements during the introduction of empagliflozin. We provide specific recommendations for special circumstances like pregnancy and liver transplantation.

Socioeconomic representativeness of Australian, Canadian and British cohorts from the paediatric diabetes AdDIT study: comparisons to regional and national data.

BACKGROUND: Given limited data regarding the involvement of disadvantaged groups in paediatric diabetes clinical trials, this study aimed to evaluate the socioeconomic representativeness of participants recruited into a multinational clinical trial in relation to regional and national type 1 diabetes reference populations. METHODS: Retrospective, cross-sectional evaluation of a subset of adolescent type 1 diabetes cardiorenal intervention trial (AdDIT) participants from Australia (n = 144), Canada (n = 312) and the UK (n = 173). Validated national measures of deprivation were used: the Index of Relative Socioeconomic Disadvantage (IRSD) 2016 (Australia), the Material Resources (MR) dimension of the Canadian Marginalisation index 2016 (Canada) and the Index of Multiple Deprivation (IMD) 2015 (UK). Representativeness was assessed by comparing the AdDIT cohort's distribution of deprivation quintiles with that of the local paediatric type 1 diabetes population (regional), and the broader type 1 diabetes population for which the trial's intervention was targeted (national). RESULTS: Recruited study cohorts from each country had higher proportions of participants with higher SES, and significant underrepresentation of lower SES, in relation to their national references. The socioeconomic make-up in Australia mirrored that of the regional population (p = 0.99). For Canada, the 2nd least deprived (p = 0.001) and the most deprived quintiles (p < 0.001) were over- and under-represented relative to the regional reference, while the UK featured higher regional and national SES bias with over-representation and under-representation from the least-deprived and most-deprived quintiles (p < 0.0001). CONCLUSIONS: Significant national differences in trial participation of low SES participants were observed, highlighting limitations in access to clinical research and the importance of reporting sociodemographic representation in diabetes clinical trials. TRIAL REGISTRATION: NCT01581476. Registered on 20 April 2012.

FluoMALDI Microscopy: Matrix Co-Crystallization Simultaneously Enhances Fluorescence and MALDI Imaging.

Here, the authors report that co-crystallization of fluorophores with matrix-assisted laser desorption/ionization (MALDI) imaging matrices significantly enhances fluorophore brightness up to 79-fold, enabling the amplification of innate tissue autofluorescence. This discovery facilitates FluoMALDI, the imaging of the same biological sample by both fluorescence microscopy and MALDI imaging. The approach combines the high spatial resolution and specific labeling capabilities of fluorescence microscopy with the inherently multiplexed, versatile imaging capabilities of MALDI imaging. This new paradigm simplifies registration by avoiding physical changes between fluorescence and MALDI imaging, allowing to image the exact same cells in tissues with both modalities. Matrix-fluorophore co-crystallization also facilitates applications with insufficient fluorescence brightness. The authors demonstrate  feasibility of FluoMALDI imaging with endogenous and exogenous fluorophores and autofluorescence-based FluoMALDI of brain and kidney tissue sections. FluoMALDI will advance structural-functional microscopic imaging in cell biology, biomedicine, and pathology.

No link between handedness and spatial navigation: evidence from over 400 000 participants in 41 countries.

There is an active debate concerning the association of handedness and spatial ability. Past studies used small sample sizes. Determining the effect of handedness on spatial ability requires a large, cross-cultural sample of participants and a navigation task with real-world validity. Here, we overcome these challenges via the mobile app Sea Hero Quest. We analysed the navigation performance from 422 772 participants from 41 countries and found no reliable evidence for any difference in spatial ability between left- and right-handers across all countries. A small but growing gap in performance appears for participants over 64 years old, with left-handers outperforming right-handers. Further analysis, however, suggests that this gap is most likely due to selection bias. Overall, our study clarifies the factors associated with spatial ability and shows that left-handedness is not associated with either a benefit or a deficit in spatial ability.

The impact of functional teat number on reproductive throughput in swine.

The objective was to evaluate the impact of functional teat number on reproductive throughput in swine. Data included 735 multiparous Landrace × Large White F1 females. Sow underlined traits consisted of total teat number (TT), functional teat number (FT), nonfunctional teat number (NFT), and number of functional mammary glands (FMG). Weaning traits were calculated for both the biological and the nurse dam. For the biological dam, litter size at weaning (LSW) included a sow's biological piglets regardless of cross-fostering. For nurse dam, number weaned (NW) included the piglets a sow weaned. For the biological dam, piglet survival (PS) was calculated as litter size at weaning / (total number born × 100). Linear regression estimates were calculated in RStudio v. 1.1.456 and variance components were estimated using GIBBS3F90. Average total number born, number born alive, TT, FT, NFT, and FMG were 14.22, 13.12, 14.43, 13.96, 0.42, and 10.7, respectively. An increase in one FT enhanced (P < 0.05) LSW by 0.32 piglets and NW by 0.33 piglets. Similarly, an increase in one FT improved (P < 0.05) PS by 1.63% and reduced (P < 0.05) preweaning mortality by 2.73%. However, an increase in one FT reduced (P < 0.05) average piglet weaning weight (WW) for biological and nurse dams by 35 and 94 g, respectively. Yet an increase in one FT enhanced (P < 0.05) litter weaning weight (LWW) for biological and nurse dams by 1.3 and 1.5 kg, respectively. Heritability estimates for TT, FT, NFT, FMG, WW, LWW, LSW, and PS were 0.25, 0.22, 0.53, 0.18, 0.21, 0.22, 0.16, and 0.18, respectively. Genetic correlation estimates between FT with TT, NFT, and FMG were 0.79, 0.09, and 0.28, respectively. Estimated genetic correlations between TT with WW, LWW, LSW, and PS were 0.37, 0.38, 0.11, and -0.19, respectively. Genetic correlation estimates between FT with WW, LWW, LSW, and PS were 0.44, 0.49, 0.39, and 0.35, respectively. Results suggest increasing functional teat number would enhance both piglet survival and reproductive throughput.

RaMALDI: Enabling simultaneous Raman and MALDI imaging of the same tissue section.

Multimodal tissue imaging techniques that integrate two complementary modalities are powerful discovery tools for unraveling biological processes and identifying biomarkers of disease. Combining Raman spectroscopic imaging (RSI) and matrix-assisted laser-desorption/ionization (MALDI) mass spectrometry imaging (MSI) to obtain fused images with the advantages of both modalities has the potential of providing spatially resolved, sensitive, specific biomolecular information, but has so far involved two separate sample preparations, or even consecutive tissue sections for RSI and MALDI MSI, resulting in images with inherent disparities. We have developed RaMALDI, a streamlined, integrated, multimodal imaging workflow of RSI and MALDI MSI, performed on a single tissue section with one sample preparation protocol. We show that RaMALDI imaging of various tissues effectively integrates molecular information acquired from both RSI and MALDI MSI of the same sample, which will drive discoveries in cell biology, biomedicine, and pathology, and advance tissue diagnostics.

Cultural determinants of the gap between self-estimated navigation ability and wayfinding performance: evidence from 46 countries.

Cognitive abilities can vary widely. Some people excel in certain skills, others struggle. However, not all those who describe themselves as gifted are. One possible influence on self-estimates is the surrounding culture. Some cultures may amplify self-assurance and others cultivate humility. Past research has shown that people in different countries can be grouped into a set of consistent cultural clusters with similar values and tendencies, such as attitudes to masculinity or individualism. Here we explored whether such cultural dimensions might relate to the extent to which populations in 46 countries overestimate or underestimate their cognitive abilities in the domain of spatial navigation. Using the Sea Hero Quest navigation test and a large sample (N = 383,187) we found cultural clusters of countries tend to be similar in how they self-rate ability relative to their actual performance. Across the world population sampled, higher self-ratings were associated with better performance. However, at the national level, higher self-ratings as a nation were not associated with better performance as a nation. Germanic and Near East countries were found to be most overconfident in their abilities and Nordic countries to be most under-confident in their abilities. Gender stereotypes may play a role in mediating this pattern, with larger national positive attitudes to male stereotyped roles (Hofstede's masculinity dimension) associated with a greater overconfidence in performance at the national level. We also replicate, with higher precision than prior studies, evidence that older men tend to overestimate their navigation skill more than other groups. These findings give insight into how culture and demographics may impact self-estimates of our abilities.

Rational Design of Highly Potent and Selective Covalent MAP2K7 Inhibitors.

The mitogen-activated protein kinase signaling cascade is conserved across eukaryotes, where it plays a critical role in the regulation of activities including proliferation, differentiation, and stress responses. This pathway propagates external stimuli through a series of phosphorylation events, which allows external signals to influence metabolic and transcriptional activities. Within the cascade, MEK, or MAP2K, enzymes occupy a molecular crossroads immediately upstream to significant signal divergence and cross-talk. One such kinase, MAP2K7, also known as MEK7 and MKK7, is a protein of great interest in the molecular pathophysiology underlying pediatric T cell acute lymphoblastic leukemia (T-ALL). Herein, we describe the rational design, synthesis, evaluation, and optimization of a novel class of irreversible MAP2K7 inhibitors. With a streamlined one-pot synthesis, favorable in vitro potency and selectivity, and promising cellular activity, this novel class of compounds wields promise as a powerful tool in the study of pediatric T-ALL.

Entropy and a sub-group of geometric measures of paths predict the navigability of an environment.

Despite extensive research on navigation, it remains unclear which features of an environment predict how difficult it will be to navigate. We analysed 478,170 trajectories from 10,626 participants who navigated 45 virtual environments in the research app-based game Sea Hero Quest. Virtual environments were designed to vary in a range of properties such as their layout, number of goals, visibility (varying fog) and map condition. We calculated 58 spatial measures grouped into four families: task-specific metrics, space syntax configurational metrics, space syntax geometric metrics, and general geometric metrics. We used Lasso, a variable selection method, to select the most predictive measures of navigation difficulty. Geometric features such as entropy, area of navigable space, number of rings and closeness centrality of path networks were among the most significant factors determining the navigational difficulty. By contrast a range of other measures did not predict difficulty, including measures of intelligibility. Unsurprisingly, other task-specific features (e.g. number of destinations) and fog also predicted navigation difficulty. These findings have implications for the study of spatial behaviour in ecological settings, as well as predicting human movements in different settings, such as complex buildings and transport networks and may aid the design of more navigable environments.

Leptotrichia Bacteremia: 10-Year Retrospective Clinical Analysis and Antimicrobial Susceptibility Profiles.

Leptotrichia species are anaerobic, Gram-negative bacilli increasingly recognized as pathogens capable of causing invasive infections such as bloodstream infection (BSI), particularly among immunocompromised patients. However, there is a paucity of data regarding epidemiology, antimicrobial susceptibility, optimal treatment, and clinical outcomes among patients with Leptotrichia bacteremia. Patient risk factors, treatment approaches, and outcomes of a retrospective cohort of adult patients with Leptotrichia BSI at a tertiary medical center (Mayo Clinic Rochester [MCR]) were evaluated. Concurrently, species, temporal trends, and antimicrobial susceptibility testing (AST) results of Leptotrichia isolates submitted to a reference laboratory (Mayo Clinic Laboratories) over the past 10 years were examined. We identified 224 blood culture isolates of Leptotrichia species, with 26 isolates from patients treated at MCR. The most frequent species included L. trevisanii (49%), L. buccalis (24%), and L. wadei (16%). Leptotrichia species demonstrated >90% susceptibility to penicillin, metronidazole, ertapenem, and piperacillin-tazobactam. However, 96% (74/77) of isolates were resistant to moxifloxacin. For patients treated at MCR, the mean patient age was 55 years (standard deviation [SD], 17), with 9 females (35%), and all were neutropenic at the time of BSI. The primary sources of infection were gastrointestinal (58%), intravascular catheter (35%), and odontogenic (15%). Patients were treated with metronidazole (42%), piperacillin-tazobactam (27%), or carbapenems (19%). The mean duration of treatment was 11 days (SD, 4.5), with a 60-day all-cause mortality of 19% and no microbiologic relapse. Leptotrichia species are rare but important causes of BSI in neutropenic patients. Due to evolving antimicrobial susceptibility profiles, a review of AST results is necessary when selecting optimal antimicrobial therapy.

Operation Moonshot: rapid translation of a SARS-CoV-2 targeted peptide immunoaffinity liquid chromatography-tandem mass spectrometry test from research into routine clinical use.

OBJECTIVES: During 2020, the UK's Department of Health and Social Care (DHSC) established the Moonshot programme to fund various diagnostic approaches for the detection of SARS-CoV-2, the pathogen behind the COVID-19 pandemic. Mass spectrometry was one of the technologies proposed to increase testing capacity. METHODS: Moonshot funded a multi-phase development programme, bringing together experts from academia, industry and the NHS to develop a state-of-the-art targeted protein assay utilising enrichment and liquid chromatography tandem mass spectrometry (LC-MS/MS) to capture and detect low levels of tryptic peptides derived from SARS-CoV-2 virus. The assay relies on detection of target peptides, ADETQALPQRK (ADE) and AYNVTQAFGR (AYN), derived from the nucleocapsid protein of SARS-CoV-2, measurement of which allowed the specific, sensitive, and robust detection of the virus from nasopharyngeal (NP) swabs. The diagnostic sensitivity and specificity of LC-MS/MS was compared with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) via a prospective study. RESULTS: Analysis of NP swabs (n=361) with a median RT-qPCR quantification cycle (Cq) of 27 (range 16.7-39.1) demonstrated diagnostic sensitivity of 92.4% (87.4-95.5), specificity of 97.4% (94.0-98.9) and near total concordance with RT-qPCR (Cohen's Kappa 0.90). Excluding Cq>32 samples, sensitivity was 97.9% (94.1-99.3), specificity 97.4% (94.0-98.9) and Cohen's Kappa 0.95. CONCLUSIONS: This unique collaboration between academia, industry and the NHS enabled development, translation, and validation of a SARS-CoV-2 method in NP swabs to be achieved in 5 months. This pilot provides a model and pipeline for future accelerated development and implementation of LC-MS/MS protein/peptide assays into the routine clinical laboratory.

Performance of creatinine-based equations to estimate glomerular filtration rate in White and Black populations in Europe, Brazil and Africa.

BACKGROUND: A new Chronic Kidney Disease Epidemiology Collaboration equation without the race variable has been recently proposed (CKD-EPIAS). This equation has neither been validated outside USA nor compared with the new European Kidney Function Consortium (EKFC) and Lund-Malmö Revised (LMREV) equations, developed in European cohorts. METHODS: Standardized creatinine and measured glomerular filtration rate (GFR) from the European EKFC cohorts (n = 13 856 including 6031 individuals in the external validation cohort), from France (n = 4429, including 964 Black Europeans), from Brazil (n = 100) and from Africa (n = 508) were used to test the performances of the equations. A matched analysis between White Europeans and Black Africans or Black Europeans was performed. RESULTS: In White Europeans (n = 9496), both the EKFC and LMREV equations outperformed CKD-EPIAS (bias of -0.6 and -3.2, respectively versus 5.0 mL/min/1.73 m², and accuracy within 30% of 86.9 and 87.4, respectively, versus 80.9%). In Black Europeans and Black Africans, the best performance was observed with the EKFC equation using a specific Q-value (= concentration of serum creatinine in healthy males and females). These results were confirmed in matched analyses, which showed that serum creatinine concentrations were different in White Europeans, Black Europeans and Black Africans for the same measured GFR, age, sex and body mass index. Creatinine differences were more relevant in males. CONCLUSION: In a European and African cohort, the performances of CKD-EPIAS remain suboptimal. The EKFC equation, using usual or dedicated population-specific Q-values, presents the best performance in the whole age range in the European and African populations included in this study.

Reported sleep duration reveals segmentation of the adult life-course into three phases.

Classically the human life-course is characterized by youth, middle age and old age. A wide range of biological, health and cognitive functions vary across this life-course. Here, using reported sleep duration from 730,187 participants across 63 countries, we find three distinct phases in the adult human life-course: early adulthood (19-33yrs), mid-adulthood (34-53yrs), and late adulthood (54+yrs). They appear stable across culture, gender, education and other demographics. During the third phase, where self-reported sleep duration increases with age, cognitive performance, as measured by spatial navigation, was found to have an inverted u-shape relationship with reported sleep duration: optimal performance peaks at 7 hours reported sleep. World-wide self-reported sleep duration patterns are geographically clustered, and are associated with economy, culture, and latitude.

Nanopore Analysis as a Tool for Studying Rapid Holliday Junction Dynamics and Analyte Binding.

Holliday junctions (HJs) are an important class of nucleic acid structure utilized in DNA break repair processes. As such, these structures have great importance as therapeutic targets and for understanding the onset and development of various diseases. Single-molecule fluorescence resonance energy transfer (smFRET) has been used to study HJ structure-fluctuation kinetics, but given the rapid time scales associated with these kinetics (approximately sub-milliseconds) and the limited bandwidth of smFRET, these studies typically require one to slow down the structure fluctuations using divalent ions (e.g., Mg2+). This modification limits the ability to understand and model the underlying kinetics associated with HJ fluctuations. We address this here by utilizing nanopore sensing in a gating configuration to monitor DNA structure fluctuations without divalent ions. A nanopore analysis shows that HJ fluctuations occur on the order of 0.1-10 ms and that the HJ remains locked in a single conformation with short-lived transitions to a second conformation. It is not clear what role the nanopore plays in affecting these kinetics, but the time scales observed indicate that HJs are capable of undergoing rapid transitions that are not detectable with lower bandwidth measurement techniques. In addition to monitoring rapid HJ fluctuations, we also report on the use of nanopore sensing to develop a highly selective sensor capable of clear and rapid detection of short oligo DNA strands that bind to various HJ targets.

Understanding the role of SGLT2 inhibitors in glycogen storage disease type Ib: the experience of one UK centre.

BACKGROUND: Glycogen storage disease type Ib (GSD Ib) is a severe disorder of carbohydrate metabolism due to bi-allelic variants in SLC37A4. It is associated with neutropaenia and neutrophil dysfunction, which has recently been attributed to the accumulation of 1,5-anhydroglucitol-6-phosphate (1,5AG6P) within neutrophils. Treatment with sodium-glucose co-transporter-2 (SGLT2) inhibitors, such as empagliflozin, is a novel therapy that reduces 1,5-anhydroglucitol (1,5AG) in plasma. RESULTS: We report our experience in treating 8 paediatric GSD Ib patients with empagliflozin with a cumulative treatment time greater than 12 years. Treatment with a median dose of 5 mg (0.22 mg/kg height weight) of empagliflozin resulted in improvement in bowel health, growth, and laboratory parameters. Plasma 1,5AG levels reduced by a median of 78%. Baseline 1,5AG levels in our cohort were higher than in adult patients with GSD Ib. Hypoglycaemia on empagliflozin treatment occurred in 50% of our cohort. CONCLUSION: We report the largest single centre cohort of GSD Ib patients treated with empagliflozin to date. Treatment with SGLT2 inhibitors is a novel and favourable treatment option for neutropaenia and neutrophil dysfunction in GSD Ib. We suggest a low starting dose of empagliflozin with careful titration due to the risk of hypoglycaemia. The interpretation of 1,5AG levels and their role in treatment monitoring is yet to be established, and requires ongoing research.

Lessons for a SECURE Future: Evaluating Diversity in Crop Biotechnology Across Regulatory Regimes.

Regulation of next-generation crops in the United States under the newly implemented "SECURE" rule promises to diversify innovation in agricultural biotechnology. Specifically, SECURE promises to expand the number of products eligible for regulatory exemption, which proponents theorize will increase the variety of traits, genes, organisms, and developers involved in developing crop biotechnology. However, few data-driven studies have looked back at the history of crop biotechnology to understand how specific regulatory pathways have affected diversity in crop biotechnology and how those patterns might change over time. In this article, we draw upon 30 years of regulatory submission data to 1) understand historical diversification trends across the landscape and history of past crop biotechnology regulatory pathways and 2) forecast how the new SECURE regulations might affect future diversification trends. Our goal is to apply an empirical approach to exploring the relationship between regulation and diversity in crop biotechnology and provide a basis for future data-driven analysis of regulatory outcomes. Based on our analysis, we suggest that diversity in crop biotechnology does not follow a single trajectory dictated by the shifts in regulation, and outcomes of SECURE might be more varied and restrictive despite the revamped exemption categories. In addition, the concept of confidential business information and its relationship to past and future biotechnology regulation is reviewed in light of our analysis.

Entropy of city street networks linked to future spatial navigation ability.

The cultural and geographical properties of the environment have been shown to deeply influence cognition and mental health1-6. Living near green spaces has been found to be strongly beneficial7-11, and urban residence has been associated with a higher risk of some psychiatric disorders12-14-although some studies suggest that dense socioeconomic networks found in larger cities provide a buffer against depression15. However, how the environment in which one grew up affects later cognitive abilities remains poorly understood. Here we used a cognitive task embedded in a video game16 to measure non-verbal spatial navigation ability in 397,162 people from 38 countries across the world. Overall, we found that people who grew up outside cities were better at navigation. More specifically, people were better at navigating in environments that were topologically similar to where they grew up. Growing up in cities with a low street network entropy (for example, Chicago) led to better results at video game levels with a regular layout, whereas growing up outside cities or in cities with a higher street network entropy (for example, Prague) led to better results at more entropic video game levels. This provides evidence of the effect of the environment on human cognition on a global scale, and highlights the importance of urban design in human cognition and brain function.

Effect of serum sample storage temperature on metabolomic and proteomic biomarkers.

Prospective biomarker studies can be used to identify biomarkers predictive of disease onset. However, if serum biomarkers are measured years after their collection, the storage conditions might affect analyte concentrations. Few data exists concerning which metabolites and proteins are affected by storage at - 20 °C vs - 80 °C. Our objectives were to document analytes affected by storage of serum samples at - 20 °C vs - 80 °C, and to identify those indicative of the storage temperature. We utilized liquid chromatography tandem mass spectrometry and Luminex to quantify 300 analytes from serum samples of 16 Finnish individuals with type 1 diabetes, with split-aliquot samples stored at - 80 °C and - 20 °C for a median of 4.2 years. Results were validated in 315 Finnish and 916 Scottish individuals with type 1 diabetes, stored at - 20 °C and at - 80 °C, respectively. After quality control, we analysed 193 metabolites and proteins of which 120 were apparently unaffected and 15 clearly susceptible to storage at - 20 °C vs - 80 °C. Further, we identified serum glutamate/glutamine ratio greater than 0.20 as a biomarker of storage at - 20 °C vs - 80 °C. The results provide a catalogue of analytes unaffected and affected by storage at - 20 °C vs - 80 °C and biomarkers indicative of sub-optimal storage.