Structure and Interactions of HIV-1 gp41 CHR-NHR Reverse Hairpin Constructs Reveal Molecular Determinants of Antiviral Activity.

Engineered reverse hairpin constructs containing a partial C-heptad repeat (CHR) sequence followed by a short loop and full-length N-heptad repeat (NHR) were previously shown to form trimers in solution and to be nanomolar inhibitors of HIV-1 Env mediated fusion. Their target is the in situ gp41 fusion intermediate, and they have similar potency to other previously reported NHR trimers. However, their design implies that the NHR is partially covered by CHR, which would be expected to limit potency. An exposed hydrophobic pocket in the folded structure may be sufficient to confer the observed potency, or they may exist in a partially unfolded state exposing full length NHR. Here we examined their structure by crystallography, CD and fluorescence, establishing that the proteins are folded hairpins both in crystal form and in solution. We examined unfolding in the milieu of the fusion reaction by conducting experiments in the presence of a membrane mimetic solvent and by engineering a disulfide bond into the structure to prevent partial unfolding. We further examined the role of the hydrophobic pocket, using a hairpin-small molecule adduct that occluded the pocket, as confirmed by X-ray footprinting. The results demonstrated that the NHR region nominally covered by CHR in the engineered constructs and the hydrophobic pocket region that is exposed by design were both essential for nanomolar potency and that interaction with membrane is likely to play a role in promoting the required inhibitor structure. The design concepts can be applied to other Class 1 viral fusion proteins.

Proton FLASH: Impact of Dose Rate and Split Dose on Acute Skin Toxicity in a Murine Model.

PURPOSE: Preclinical studies have shown a preferential normal tissue sparing effect of FLASH radiation therapy with ultra-high dose rates. The aim of the present study was to use a murine model of acute skin toxicity to investigate the biologic effect of varying dose rates, time structure, and introducing pauses in the dose delivery. METHODS AND MATERIALS: The right hind limbs of nonanaesthetized mice were irradiated in the entrance plateau of a pencil beam scanning proton beam with 39.3 Gy. Experiment 1 was with varying field dose rates (0.7-80 Gy/s) without repainting, experiment 2 was with varying field dose rates (0.37-80 Gy/s) with repainting, and in experiment 3, the dose was split into 2, 3, 4, or 6 identical deliveries with 2-minute pauses. In total, 320 mice were included, with 6 to 25 mice per group. The endpoints were skin toxicity of different levels up to 25 days after irradiation. RESULTS: The dose rate50, which is the dose rate to induce a response in 50% of the animals, depended on the level of skin toxicity, with the higher toxicity levels displaying a FLASH effect at 0.7-2 Gy/s. Repainting resulted in higher toxicity for the same field dose rate. Splitting the dose into 2 deliveries reduced the FLASH effect, and for 3 or more deliveries, the FLASH effect was almost abolished for lower grades of toxicity. CONCLUSIONS: The dose rate that induced a FLASH effect varied for different skin toxicity levels, which are characterized by a differing degree of sensitivity to radiation dosage. Conclusions on a threshold for the dose rate needed to obtain a FLASH effect can therefore be influenced by the dose sensitivity of the used endpoint. Splitting the total dose into more deliveries compromised the FLASH effect. This can have an impact for fractionation as well as for regions where 2 or more FLASH fields overlap within the same treatment session.

Two-dimensional time-resolved scintillating sheet monitoring of proton pencil beam scanning FLASH mouse irradiations.

BACKGROUND: Dosimetry in pre-clinical FLASH studies is essential for understanding the beam delivery conditions that trigger the FLASH effect. Resolving the spatial and temporal characteristics of proton pencil beam scanning (PBS) irradiations with ultra-high dose rates (UHDR) requires a detector with high spatial and temporal resolution. PURPOSE: To implement a novel camera-based system for time-resolved two-dimensional (2D) monitoring and apply it in vivo during pre-clinical proton PBS mouse irradiations. METHODS: Time-resolved 2D beam monitoring was performed with a scintillation imaging system consisting of a 1 mm thick transparent scintillating sheet, imaged by a CMOS camera. The sheet was placed in a water bath perpendicular to a horizontal PBS proton beam axis. The scintillation light was reflected through a system of mirrors and captured by the camera with 500 frames per second (fps) for UHDR and 4 fps for conventional dose rates. The raw images were background subtracted, geometrically transformed, flat field corrected, and spatially filtered. The system was used for 2D spot and field profile measurements and compared to radiochromic films. Furthermore, spot positions were measured for UHDR irradiations. The measured spot positions were compared to the planned positions and the relative instantaneous dose rate to equivalent fiber-coupled point scintillator measurements. For in vivo application, the scintillating sheet was placed 1 cm upstream the right hind leg of non-anaesthetized mice submerged in the water bath. The mouse leg and sheet were both placed in a 5 cm wide spread-out Bragg peak formed from the mono-energetic proton beam by a 2D range modulator. The mouse leg position within the field was identified for both conventional and FLASH irradiations. For the conventional irradiations, the mouse foot position was tracked throughout the beam delivery, which took place through repainting. For FLASH irradiations, the delivered spot positions and relative instantaneous dose rate were measured. RESULTS: The pixel size was 0.1 mm for all measurements. The spot and field profiles measured with the scintillating sheet agreed with radiochromic films within 0.4 mm. The standard deviation between measured and planned spot positions was 0.26 mm and 0.35 mm in the horizontal and vertical direction, respectively. The measured relative instantaneous dose rate showed a linear relation with the fiber-coupled scintillator measurements. For in vivo use, the leg position within the field varied between mice, and leg movement up to 3 mm was detected during the prolonged conventional irradiations. CONCLUSIONS: The scintillation imaging system allowed for monitoring of UHDR proton PBS delivery in vivo with 0.1 mm pixel size and 2 ms temporal resolution. The feasibility of instantaneous dose rate measurements was demonstrated, and the system was used for validation of the mouse leg position within the field.

Oxygen Enhancement Ratio-Weighted Dose Quantitatively Describes Acute Skin Toxicity Variations in Mice After Pencil Beam Scanning Proton FLASH Irradiation With Changing Doses and Time Structures.

PURPOSE: The aim of this work was to investigate the ability of a biological oxygen enhancement ratio-weighted dose, DOER, to describe acute skin toxicity variations observed in mice after proton pencil beam scanning irradiations with changing doses and beam time structures. METHODS AND MATERIALS: In five independent experiments, the right hind leg of a total of 621 CDF1 mice was irradiated previously in the entrance plateau of a pencil beam scanning proton beam. The incidence of acute skin toxicity (of level 1.5-2.0-2.5-3.0-3.5) was scored for 47 different mouse groups that mapped toxicity as function of dose for conventional and FLASH dose rate, toxicity as function of field dose rate with and without repainting, and toxicity when splitting the treatment into 1 to 6 identical deliveries separated by 2 minutes. DOER was calculated for all mouse groups using a simple oxygen kinetics model to describe oxygen depletion. The three independent model parameters (oxygen-depletion rate, oxygen-recovery rate, oxygen level without irradiation) were fitted to the experimental data. The ability of DOER to describe the toxicity variations across all experiments was investigated by comparing DOER-response curves across the five independent experiments. RESULTS: After conversion from the independent variable tested in each experiment to DOER, all five experiments had similar MDDOER50 (DOER giving 50% toxicity incidence) with standard deviations of 0.45 - 1.6 Gy for the five toxicity levels. DOER could thus describe the observed toxicity variations across all experiments. CONCLUSIONS: DOER described the varying FLASH-sparing effect observed for a wide range of conditions. Calculation of DOER for other irradiation conditions can quantitatively estimate the FLASH-sparing effect for arbitrary irradiations for the investigated murine model. With appropriate fitting parameters DOER also may be able to describe FLASH effect variations with dose and dose rate for other assays and endpoints.

Not all grammar errors are equally noticed: error detection of naturally occurring errors and implications for eye-tracking models of everyday texts.

Grammar errors are a natural part of everyday written communication. They are not a uniform group, but vary from morphological errors to ungrammatical word order and involve different types of word classes. In this study, we examine whether some types of naturally occurring errors attract more attention than others during reading, measured by detection rates. Data from 211 Danish high school students were included in the analysis. They each read texts containing different types of errors: syntactic errors (verb-third word order), morphological agreement errors (verb conjugations; gender mismatches in NPs) and orthographic errors. Participants were asked to underline all errors they detected while reading for comprehension. We examined whether there was a link between the type of errors that participants did not detect, the type of errors which they produce themselves (as measured in a subsequent grammar quiz), and the type of errors that are typical of high school students in general (based on error rates in a corpus). If an error is infrequent in production, it may cause a larger surprisal effect and be more attended to. For the three subtypes of grammar errors (V3 word order, verb errors, NP errors), corpus error rates predicted detection rates for most conditions. Yet, frequency was not the only possible explanation, as phonological similarity to the correct form is entangled with error frequency. Explicit grammatical awareness also played a role. The more correct answers participants had in the grammar tasks in the quiz, the more errors they detected. Finally, we found that the more annoyed with language errors participants reported to be, the more errors they detected. Our study did not measure eye movements, but the differences in error detection patterns point to shortcomings of existing eye-tracking models. Understanding the factors that govern attention and reaction to everyday grammar errors is crucial to developing robust eye-tracking processing models which can accommodate non-standard variation. Based on our results, we give our recommendations for current and future processing models.

Major Cardiovascular Events After Spontaneous Intracerebral Hemorrhage by Hematoma Location.

Importance: Survivors of spontaneous (ie, nontraumatic and with no known structural cause) intracerebral hemorrhage (ICH) have an increased risk of major cardiovascular events (MACEs), including recurrent ICH, ischemic stroke (IS), and myocardial infarction (MI). Only limited data are available from large, unselected population studies assessing the risk of MACEs according to index hematoma location. Objective: To examine the risk of MACEs (ie, the composite of ICH, IS, spontaneous intracranial extra-axial hemorrhage, MI, systemic embolism, or vascular death) after ICH based on ICH location (lobar vs nonlobar). Design, Setting, and Participants: This cohort study identified 2819 patients in southern Denmark (population of 1.2 million) 50 years or older hospitalized with first-ever spontaneous ICH from January 1, 2009, to December 31, 2018. Intracerebral hemorrhage was categorized as lobar or nonlobar, and the cohorts were linked to registry data until the end of 2018 to identify the occurrence of MACEs and separately recurrent ICH, IS, and MI. Outcome events were validated using medical records. Associations were adjusted for potential confounders using inverse probability weighting. Exposure: Location of ICH (lobar vs nonlobar). Main Outcomes and Measures: The main outcomes were MACEs and separately recurrent ICH, IS, and MI. Crude absolute event rates per 100 person-years and adjusted hazard ratios (aHRs) with 95% CIs were calculated. Data were analyzed from February to September 2022. Results: Compared with patients with nonlobar ICH (n = 1255; 680 [54.2%] men and 575 [45.8%] women; mean [SD] age, 73.5 [11.4] years), those with lobar ICH (n = 1034; 495 [47.9%] men and 539 [52.1%] women, mean [SD] age, 75.2 [10.7] years) had higher rates of MACEs per 100 person-years (10.84 [95% CI, 9.51-12.37] vs 7.91 [95% CI, 6.93-9.03]; aHR, 1.26; 95% CI, 1.10-1.44) and recurrent ICH (3.74 [95% CI, 3.01-4.66] vs 1.24 [95% CI, 0.89-1.73]; aHR, 2.63; 95% CI, 1.97-3.49) but not IS (1.45 [95% CI, 1.02-2.06] vs 1.77 [95% CI, 1.34-2.34]; aHR, 0.81; 95% CI, 0.60-1.10) or MI (0.42 [95% CI, 0.22-0.81] vs 0.64 [95% CI, 0.40-1.01]; aHR, 0.64; 95% CI, 0.38-1.09). Conclusions and Relevance: In this cohort study, spontaneous lobar ICH was associated with a higher rate of subsequent MACEs than nonlobar ICH, primarily due to a higher rate of recurrent ICH. This study highlights the importance of secondary ICH prevention strategies in patients with lobar ICH.

Effect of atropine and propofol on the minimum anaesthetic concentration of isoflurane in the freshwater turtle Trachemys scripta (yellow-bellied slider).

OBJECTIVE: To determine if the administration of atropine would reduce the measured minimum anaesthetic concentration of isoflurane (MACisoflurane) in freshwater turtles - the yellow-bellied slider (Trachemys scripta scripta). STUDY DESIGN: Paired, blinded, randomized, prospective studies of 1) the effect of atropine in isoflurane anaesthetized freshwater turtles (T. scripta scripta) and 2) the effect of atropine in yellow-bellied sliders in which anaesthesia was induced with propofol and maintained with isoflurane. ANIMALS: T. scripta scripta (n = 8), female, adult. METHODS: Atropine (2 mg kg-1) or an isovolumetric control injection of saline was administered intraperitoneally 15 minutes prior to induction of anaesthesia with isoflurane. Individual MACisoflurane was then determined by end-tidal gas analysis in a bracketing design by an experimenter blinded to the administered drug, with a 2 week washout period. The experiment was repeated, with atropine (2 mg kg-1) or saline administered intravascularly in combination with propofol for anaesthetic induction. Linear mixed modelling was used to determine the effects of atropine and propofol on the individual MACisoflurane. Data are presented as mean ± standard deviation. RESULTS: Premedication with atropine significantly reduced MACisoflurane (p = 0.0039). In isoflurane-induced T. scripta scripta, MACisoflurane decreased from 4.2 ± 0.4% to 3.3 ± 0.8% when atropine had been administered. Propofol as an induction agent had a MAC-sparing effect (p < 0.001) such that MACisoflurane following propofol and a control injection of saline was 2.3 ± 1.0%, which decreased further to 1.5 ± 0.8% when propofol was combined with atropine. CONCLUSIONS AND CLINICAL RELEVANCE: Atropine, presumably by inhibiting parasympathetically mediated pulmonary artery constriction, decreases right-to-left cardiac shunting and the MACisoflurane in yellow-bellied sliders, and thereby may facilitate control of inhalant anaesthesia. Propofol can be used for induction of anaesthesia and reduces the required concentration of inhaled anaesthesia assessed 1.5 hours following induction.

Beta cell endoplasmic reticulum stress drives diabetes in the KINGS mouse without causing mass beta cell loss.

AIMS: Beta cell endoplasmic reticulum (ER) stress can cause cellular death and dysfunction and has been implicated in the pathogenesis of diabetes. Animal models of beta cell ER stress are critical in further understanding this and for testing novel diabetes therapeutics. The KINGS mouse is a model of beta cell ER stress driven by a heterozygous mutation in Ins2. In this study, we investigated how beta cell ER stress in the KINGS mouse drives diabetes. METHODS: We investigated whether the unfolded protein response (UPR) was activated in islets isolated from male and female KINGS mice and whether this impacted beta cell mass and turnover. RESULTS: Whilst the UPR was up-regulated in KINGS islets, with increased protein expression of markers of all three UPR arms, this was not associated with a mass loss of beta cells; beta cell apoptosis rates did not increase until after the development of overt diabetes, and did not lead to substantial changes in beta cell mass. CONCLUSION: We propose that the KINGS mouse represents a model where beta cell maladaptive UPR signalling drives diabetes development without causing mass beta cell loss.

An automated liquid jet for fluorescence dosimetry and microsecond radiolytic labeling of proteins.

X-ray radiolytic labeling uses broadband X-rays for in situ hydroxyl radical labeling to map protein interactions and conformation. High flux density beams are essential to overcome radical scavengers. However, conventional sample delivery environments, such as capillary flow, limit the use of a fully unattenuated focused broadband beam. An alternative is to use a liquid jet, and we have previously demonstrated that use of this form of sample delivery can increase labeling by tenfold at an unfocused X-ray source. Here we report the first use of a liquid jet for automated inline quantitative fluorescence dosage characterization and sample exposure at a high flux density microfocused synchrotron beamline. Our approach enables exposure times in single-digit microseconds while retaining a high level of side-chain labeling. This development significantly boosts the method's overall effectiveness and efficiency, generates high-quality data, and opens up the arena for high throughput and ultrafast time-resolved in situ hydroxyl radical labeling.

Obesity prolongs induction times in reptiles.

Obesity is common in captive reptiles, and reptiles are increasingly popular as companion animals and in physiological research. Obesity may present a challenge during surgical procedures using inhalation anaesthesia, as the long induction time due to the low reptilian metabolism may increase anaesthetic accumulation in the adipose tissues. This study investigated the impact of obesity on induction and recovery times from inhaled anaesthesia. The temporal change in the partial pressure of isoflurane in different tissues was predicted using a multi-compartment model. Furthermore, as right-to-left shunting can delay anaesthetic uptake and washout, we included an assessment of the combination of cardiac shunting and obesity. The model predictions indicate a clear increase in time to reach 90% equilibration of administered anaesthetic in the brain (T90) of obese non-shunting (lean 47 min, obese >100 min) and shunting (lean 81 min, obese >100 min) reptiles. The combination of obesity and shunting doubled the time to acquisition of mean anaesthetic concentration (a measure used to plan anaesthesia) from 8 min to 19 min. Adipose blood flow highly affected whether the body type had an impact on induction time, with low adipose blood flow abolishing the effect of body type. As T90 was never reached within 100 min with both the obese reptiles, it was not possible to conclude on the effect of obesity on recovery times within this study. Care should therefore be taken when anaesthetising obese reptiles for surgical purposes, to ensure adequate anaesthetic depth is attained, and recovery monitored closely.

Validity of Simple Algorithms to Identify Recurrence of Intracerebral Hemorrhage in Two Danish Nationwide Registries.

PURPOSE: Danish registries could be an attractive resource for studies of recurrent intracerebral hemorrhage (re-ICH). We developed and validated algorithms to identify re-ICH in the Danish Stroke Registry (DSR) and the Danish National Patient Registry (DNPR). PATIENTS AND METHODS: Using multiple sources, we followed-up an inception cohort with verified first-ever spontaneous ICH (n = 2528) for their first re-ICH in 2009-2018 (study period). We used verified cases of re-ICH (n = 124) as the gold standard to assess the performance of register-based algorithms for identifying re-ICH. For each cohort member, we traced events of re-ICH (ICD-10-code I61) in the study period according to DSR and DNPR, respectively. For each registry, we tested algorithms with a blanking period (BP) - ie, a period immediately following the index ICH during which outcome events were ignored - of varying length (7 days-360 days). The algorithm with the shortest BP that returned a positive predictive value (PPV) of ≥80% was considered optimal. We also calculated negative predictive value (NPV), sensitivity, and specificity of each algorithm and [95% confidence intervals] for all proportions. RESULTS: The optimal algorithm for DSR (BP 30 days) had a PPV of 89.5% [82.2-94.0], NPV 98.8% [98.2-99.1], sensitivity 75.8% [67.6-82.5], and specificity 99.5% [99.2-99.7]. The optimal algorithm for DNPR (BP 120 days) had a PPV of 80.6% [71.7-87.2], NPV 98.1% [97.5-98.6], sensitivity 63.7% [55.0-71.6], and specificity 99.2% [98.8-99.5]. CONCLUSION: Simple algorithms accurately identified re-ICH in DSR and DNPR. Compared with DNPR, DSR achieved higher PPV and sensitivity with a shorter BP. The proposed algorithms could facilitate valid use of DSR and DNPR for studies of re-ICH.

Hydroxyl radical mediated damage of proteins in low oxygen solution investigated using X-ray footprinting mass spectrometry.

In the method of X-ray footprinting mass spectrometry (XFMS), proteins at micromolar concentration in solution are irradiated with a broadband X-ray source, and the resulting hydroxyl radical modifications are characterized using liquid chromatography mass spectrometry to determine sites of solvent accessibility. These data are used to infer structural changes in proteins upon interaction with other proteins, folding, or ligand binding. XFMS is typically performed under aerobic conditions; dissolved molecular oxygen in solution is necessary in many, if not all, the hydroxyl radical modifications that are generally reported. In this study we investigated the result of X-ray induced modifications to three different proteins under aerobic versus low oxygen conditions, and correlated the extent of damage with dose calculations. We observed a concentration-dependent protecting effect at higher protein concentration for a given X-ray dose. For the typical doses used in XFMS experiments there was minimal X-ray induced aggregation and fragmentation, but for higher doses we observed formation of covalent higher molecular weight oligomers, as well as fragmentation, which was affected by the amount of dissolved oxygen in solution. The higher molecular weight products in the form of dimers, trimers, and tetramers were present in all sample preparations, and, upon X-ray irradiation, these oligomers became non-reducible as seen in SDS-PAGE. The results provide an important contribution to the large body of X-ray radiation damage literature in structural biology research, and will specifically help inform the future planning of XFMS, and well as X-ray crystallography and small-angle X-ray scattering experiments.

Food limitation and starvation independently affect predator macronutrient selection.

Predators are often food limited in their habitat, and some are limited by specific macronutrients (protein, lipid or carbohydrate). It is unresolved, however, to what extent and in what way food and macronutrient limitation are connected. Using a carabid beetle (Nebria brevicollis), we compared macronutrient self-selection of the animals three times: immediately after collection in the field, after being fed to satiation and nutritional balance and after a subsequent period of starvation. Both sexes were food and females lipid limited in the field; after 7-21 days of starvation both sexes increased proportional carbohydrate intake significantly. Thus, starvation created a nutrient deficit that was different from what the animals had experienced in the field. We conclude that while macronutrient limitation in nature may be influenced by hunger due to food limitation, this is not its main determinant. A nutritional imbalance of available food may override this effect.

A greater awareness of children with glucose-6-phosphate dehydrogenase deficiency is imperative in western countries.

AIM: This national retrospective Danish study described the characteristics of children diagnosed with glucose-6-phosphate dehydrogenase (G6PD) deficiency, an inherited X-linked recessive disorder that often affects children of Middle Eastern descent. METHODS: We studied children born between 1 January 2000 and 31 December 2017 and diagnosed with G6PD deficiency. They were identified from the Danish National Hospital Discharge Register and the Danish Database of Extreme Neonatal Hyperbilirubinaemia. RESULTS: There were 113 children diagnosed with G6PD deficiency, 67% were of Middle Eastern descent and they were frequently diagnosed before the onset of symptoms, based on known heredity. Of the 67 infants born in Denmark, 10% had extreme neonatal hyperbilirubinaemia and one developed kernicterus spectrum disorder, as did one child born in the Middle East. Most (61%) of the 33 children with jaundice received phototherapy, 12% had exchange transfusions and 18% received whole blood transfusions. After the neonatal period, 23% of the cohort had blood transfusions and 4% needed intensive care for acute haemolytic anaemia. The incidence of G6PD deficiency appeared to be severely underestimated. CONCLUSION: Many families from countries where G6PD deficiency is endemic move to Denmark and other Western countries. Greater awareness is essential to avoid chronic and potentially lethal, consequences.

The Role of Non-Selective TNF Inhibitors in Demyelinating Events.

The use of non-selective tumor necrosis factor (TNF) inhibitors is well known in the treatment of inflammatory diseases such as rheumatoid arthritis, Crohn's disease, and psoriasis. Its use in neurological disorders is limited however, due to rare adverse events of demyelination, even in patients without preceding demyelinating disease. We review here the molecular and cellular aspects of this neuroinflammatory process in light of a case of severe monophasic demyelination caused by treatment with infliximab. Focusing on the role of TNF, we review the links between CNS inflammation, demyelination, and neurodegenerative changes leading to permanent neurological deficits in a young woman, and we discuss the growing evidence for selective soluble TNF inhibitors as a new treatment approach in inflammatory and neurological diseases.

Standardization of the Prosody in Use Battery (PUB): a speech prosody perception test in Danish.

Assessment of prosody perception may be useful in a number of clinical scenarios, including the rehabilitation of cochlear implant recipients. It is with this group in mind that we have derived and standardized a battery of tests that assess speech prosody perception in the Danish language. The prosodic contrasts included in the battery are vowel length, compounds and phrases, emotions, questions and statements, prominence and pronoun reference, all of which are commonly encountered in everyday communication. Lists of candidate stimuli were compiled and recorded by a representative speaker of Danish. All candidate stimuli were presented to normal hearing subjects (n = 12) in both unprocessed and 8-channel noise vocoded conditions. Subjects performed closed-set identification and the results were used to derive the final stimulus set. We report the results of the six subtests, in which we observed a bias to compounds in the compound/phrase subtest, and to statements in question/statement subtest. The pronoun reference subtest assessed the ability of a listener to infer a referent from the stress status of a pronoun, and we found high accuracy rates on this task indicating that it is suitable for inclusion in the battery. We discuss the possible uses of the Prosody in Use Battery in cochlear implant mapping and device verification. We also consider the role of the results from the test battery in guiding clinicians to material suitable for aural rehabilitation.

Laboratory Evolution of a Saccharomyces cerevisiae × S. eubayanus Hybrid Under Simulated Lager-Brewing Conditions.

Saccharomyces pastorianus lager-brewing yeasts are domesticated hybrids of S. cerevisiae x S. eubayanus that display extensive inter-strain chromosome copy number variation and chromosomal recombinations. It is unclear to what extent such genome rearrangements are intrinsic to the domestication of hybrid brewing yeasts and whether they contribute to their industrial performance. Here, an allodiploid laboratory hybrid of S. cerevisiae and S. eubayanus was evolved for up to 418 generations on wort under simulated lager-brewing conditions in six independent sequential batch bioreactors. Characterization of 55 single-cell isolates from the evolved cultures showed large phenotypic diversity and whole-genome sequencing revealed a large array of mutations. Frequent loss of heterozygosity involved diverse, strain-specific chromosomal translocations, which differed from those observed in domesticated, aneuploid S. pastorianus brewing strains. In contrast to the extensive aneuploidy of domesticated S. pastorianus strains, the evolved isolates only showed limited (segmental) aneuploidy. Specific mutations could be linked to calcium-dependent flocculation, loss of maltotriose utilization and loss of mitochondrial activity, three industrially relevant traits that also occur in domesticated S. pastorianus strains. This study indicates that fast acquisition of extensive aneuploidy is not required for genetic adaptation of S. cerevisiae × S. eubayanus hybrids to brewing environments. In addition, this work demonstrates that, consistent with the diversity of brewing strains for maltotriose utilization, domestication under brewing conditions can result in loss of this industrially relevant trait. These observations have important implications for the design of strategies to improve industrial performance of novel laboratory-made hybrids.

Molecular architecture of the Jumonji C family histone demethylase KDM5B.

The full length human histone 3 lysine 4 demethylase KDM5B (PLU-1/Jarid1B) has been studied using Hydrogen/Deuterium exchange mass spectrometry, homology modelling, sequence analysis, small angle X-ray scattering and electron microscopy. This first structure on an intact multi-domain Jumonji histone demethylase reveal that the so-called PLU region, in the central region of KDM5B, has a curved α-helical three-dimensional structure, that acts as a rigid linker between the catalytic core and a region comprising four α-helices, a loop comprising the PHD2 domain, two large intrinsically disordered loops and the PHD3 domain in close proximity. The dumbbell shaped and curved KDM5B architecture observed by electron microscopy is complementary to the nucleosome surface and has a striking overall similarity to that of the functionally related KDM1A/CoREST complex. This could suggest that there are similarities between the demethylation mechanisms employed by the two histone 3 lysine 4 demethylases at the molecular level.

Peptides Derived from Histone 3 and Modified at Position 18 Inhibit Histone Demethylase KDM6 Enzymes.

The KDM6 subfamily of histone lysine demethylases has recently been implicated as a putative target in the treatment of a number of diseases; this makes the availability of potent and selective inhibitors important. Due to high sequence similarity of the catalytic domain of Jumonji C histone demethylases, the development of small-molecule, family-specific inhibitors has, however, proven challenging. One approach to achieve the selective inhibition of these enzymes is the use of peptides derived from the substrate, the histone 3 C terminus. Here we used computational methods to optimize such inhibitors of the KDM6 family. Through natural amino acid substitution, it is shown that a K18I variant of a histone H3 derived peptide significantly increases affinity towards the KDM6 enzymes. The crystal structure of KDM6B in complex with a histone 3 derived K18I peptide reveals a tighter fit of the isoleucine side chain, compared with that of the arginine. As a consequence, the peptide R17 residue also has increased hydrophilic interactions. These interactions of the optimized peptide are likely to be responsible for the increased affinity to the KDM6 enzymes.

Mass spectrometry analyses of normal and polyglutamine expanded ataxin-3 reveal novel interaction partners involved in mitochondrial function.

Deubiquitinating enzymes (DUBs) play important roles in a variety of cellular processes, including regulation of protein homeostasis. The DUB ataxin-3 is an enzyme implicated in protein quality control mechanisms. In the neurodegenerative disease spinocerebellar ataxia type 3 (SCA3), ataxin-3 contains an expanded polyglutamine (polyQ) stretch that leads to aggregation of the protein and neuronal dysfunction. Increasing the understanding of ataxin-3 protein interaction partners could help to elucidate disease mechanisms. Hence, we analyzed the repertoire of proteins interacting with normal and polyQ expanded ataxin-3 by mass spectrometry. This showed that both normal and polyQ expanded ataxin-3 interacted with components of the protein quality control system and mitochondria. Five proteins showed increased interaction with polyQ expanded ataxin-3 relative to normal and three of these were mitochondrial proteins. The analyses underline the role of ataxin-3 in ubiquitin biology and point towards a role in mitochondrial biology.