Following the work of Day & Hawthorne [Acta Cryst. (2022), A78, 212–233] and Day et al. [Acta Cryst. (2024), A80, 258–281], the program GraphT–T has been developed to embed graphical representations of observed and hypothetical chains of (SiO4)4− tetrahedra into 2D and 3D Euclidean space. During embedding, the distance between linked vertices (T–T distances) and the distance between unlinked vertices (T⋯T separations) in the resultant unit-distance graph are restrained to the average observed distance between linked Si tetrahedra (3.06±0.15 Å) and the minimum separation between unlinked vertices is restrained to be equal to or greater than the minimum distance between unlinked Si tetrahedra (3.713 Å) in silicate minerals. The notional interactions between vertices are described by a 3D spring-force algorithm in which the attractive forces between linked vertices behave according to Hooke's law and the repulsive forces between unlinked vertices behave according to Coulomb's law. Embedding parameters (i.e. spring coefficient, k, and Coulomb's constant, K) are iteratively refined during embedding to determine if it is possible to embed a given graph to produce a unit-distance graph with T–T distances and T⋯T separations that are compatible with the observed T–T distances and T⋯T separations in crystal structures. The resultant unit-distance graphs are denoted as compatible and may form crystal structures if and only if all distances between linked vertices (T–T distances) agree with the average observed distance between linked Si tetrahedra (3.06±0.15 Å) and the minimum separation between unlinked vertices is equal to or greater than the minimum distance between unlinked Si tetrahedra (3.713 Å) in silicate minerals. If the unit-distance graph does not satisfy these conditions, it is considered incompatible and the corresponding chain of tetrahedra is unlikely to form crystal structures. Using GraphT–T, Day et al. [Acta Cryst. (2024), A80, 258–281] have shown that several topological properties of chain graphs influence the flexibility (and rigidity) of the corresponding chains of Si tetrahedra and may explain why particular compatible chain arrangements (and the minerals in which they occur) are more common than others and/or why incompatible chain arrangements do not occur in crystals despite being topologically possible.

Ganesh Betageri, MBA, serves as the head of engineering at Breach Insurance

Sparkswelll was built to support online fundraising, communication with supporters, event scheduling, email campaigns, supporter data management and field canvassing. The platform also supports federal election financial reporting requirements.

KTD BIOLABS Launches American Dietary Supplements: BeTall Height Growth Maximizer and Height Growth MAX+

Great Tibet Tour Announces Booming Tibet Tourism

Today we release our first self-hosted Auphonic Speech Recognition Engine using the open-source Whisper model by OpenAI!
With Whisper, you can now integrate automatic speech recognition in 99 languages into your Auphonic audio post-production workflow, without creating an external account and without extra costs!

Whisper Speech Recognition in Auphonic

So far, Auphonic users had to choose one of our integrated external service providers (Wit.ai, Google Cloud Speech, Amazon Transcribe, Speechmatics) for speech recognition, so audio files were transferred to an external server, using external computing powers, that users had to pay for in their external accounts.

The new Auphonic Speech Recognition is using Whisper, which was published by OpenAI as an open-source project. Open-source means, the publicly shared GitHub repository contains a complete Whisper package including source code, examples, and research results.
However, automatic speech recognition is a very time and hardware-consuming process, that can be incredibly slow using a standard home computer without special GPUs. So we decided to integrate this service and offer you automatic speech recognition (ASR) by Whisper processed on our own hardware, just like any other Auphonic processing task, giving you quite some benefits:

• No external account is needed anymore to run ASR in Auphonic.
• Your data doesn't leave our Auphonic servers for ASR processing.
• No extra costs for external ASR services.
• Additional Auphonic pre- and post-processing for more accurate ASR, especially for Multitrack Productions.
• The quality of Whisper ASR is absolutely comparable to the “best” services in our comparison table.

How to use Whisper?

To use the Auphonic Whisper integration, you just have to create a production or preset as you are used to and select “Auphonic Whisper ASR” as “Service” in the section Speech Recognition.
This option will automatically appear for Beta and paying users. If you are a free user but want to try Whisper: please just ask for access!

When your Auphonic speech recognition is done, you can download your transcript in different formats and may edit or share your transcript with the Auphonic Transcript Editor.
For more details about all our integrated speech recognition services, please visit our Speech Recognition Help and watch this channel for Whisper updates – soon to come.

Why Beta?

We decided to launch Whisper for Beta and paying users only, as Whisper was just published end of September and there was not enough time to test every single use case sufficiently.
Another issue is the required computing power: for suitable scaling of the GPU infrastructure, we need a beta phase to test the service while we are monitoring the hardware usage, to make sure there are no server overloads.

Conclusion

Automatic speech recognition services are evolving very quickly, and we've seen major improvements over the past few years.
With Whisper, we can now perform speech recognition without extra costs on our own GPU hardware, no external services are required anymore.

Auphonic Whisper ASR is available for Beta and paying users now, free users can ask for Beta access.
You are very welcome to send us feedback (directly in the production interface or via email), whether you notice something that works particularly well or discover any problems.
Your feedback is a great help to improve the system!

In addition to our Leveler, Denoiser, and Adaptive 'Hi-Pass' Filter, we now release the missing equalization feature with the new Auphonic AutoEQ.
The AutoEQ automatically analyzes and optimizes the frequency spectrum of a voice recording, to remove sibilance (De-esser) and to create a clear, warm, and pleasant sound - listen to the audio examples below to get an idea about what it does.

Screenshot of manually adjusted example settings for the equalizer plug-in 'Pro-Q3' by fabfilter.

What is Equalization and why is it difficult?

Equalization (EQ) in audio recording and reproduction is the process of adjusting the volume of different frequency bands within a signal.
The following vocal EQ cheat sheet, published by Producer Hive, will give you a small impression, of what can be influenced by equalizing:

Vocal EQ Cheat Sheet by Producer Hive.

On the other hand, it is very easy to ruin a good voice recording with manual heavy-handed equalization, resulting in voices, that sound very sharp or muddy or even like the speaker had a blocked nose.
Besides the skill and experience of an audio engineer, manual adjustments of frequencies also require a very good and linear studio playback device. For example, performing manual equalization with strongly bass-heavy speakers would most likely lead to a very sharp, unpleasant listening experience using treble-heavy headphones.

For singletrack productions with more than one speaker, equalizing is also a very complex and time-consuming process, as every voice has its unique frequency spectrum and needs its own equalization. One could separate speakers with cuts or create a track envelope to fade from one speaker to another, however, any solution is a very tedious job, if you do it by hand.

That is where the Auphonic AutoEQ comes in! All those steps are now available in just one click!

How does the Auphonic AutoEQ work?

The Auphonic Web Service analyzes your audio content and classifies the audio file into small and meaningful segments like music, silence, different speakers, etc. to process every single segment with the best matching algorithms.
All our features like the Adaptive Leveler, Dynamic Denoising, Adaptive 'Hi-Pass' Filtering, and now the new AutoEQ filter option are built on top of this basic processing.

Using Auphonic AutoEQ, spectral EQ profiles are created for each speaker separately and permanently changing over time. The aim of those time-dependent EQ profiles is to create a constant, pleasant sound in the output file even if there are slightly changing voices in the record, for example, due to modified speaker-microphone positions.

Audio Examples

Here are two short audio examples, which demonstrate some features of our AutoEQ.
We recommend listening with headphones so you can hear all the details.

Example 1. Female Speaker with Background Music

In the following example (BCB: The Voices of Bainbridge Island) of a female narrator speaking while background music is playing, you can easily recognize quite sharp 'sss' sounds in the female voice. This sharpness in the female voice is removed by the so-called De-essing feature of the Auphonic AutoEQ, while the background music is not changed.

Original:
AutoEQed:

Example 2. Dialog of Male and Female Speakers

The next example (BCB: The Voices of Bainbridge Island) shows how the AutoEQ optimizes a singletrack record containing two speakers with different voice characteristics. Our AutoEQ algorithms analyze each voice separately and calculate the matching frequency adjustments to optimize the voice of every single speaker.

Original:
AutoEQed:

AutoEQ Beta Integration in the Auphonic Web Service

To use the Auphonic AutoEQ, you just have to create a production or preset as you are used to, toggle “Advanced Parameters” on the top right in the section “Audio Algorithms” and select “Voice AutoEQ” within “Filtering”:

For a first test period, the AutoEQ will only be available for Beta and paying users, to incorporate your feedback and finalize an optimized version.
If you are a free user but want to try Auphonic AutoEQ: please just ask for access!

Practical Tips

For best results using Auphonic AutoEQ, however, it is still necessary, that your audio content is of sufficiently good quality, as no equalizer can make up frequencies that are not there in the first place. Audio files with low bitrates often lack important frequencies, that cannot be recovered by equalizing. AutoEQ is just a feature to boost or cut individual frequency bands, not a bandwidth extension. For more information about required audio quality, see the former blog post: Audio File Formats and Bitrates for Podcasts.

Another important topic is the definition of the 'best result'. Equalizing is a very subjective task, that differs a lot depending on every personal opinion. So Auphonic AutoEQ is set up to follow quite conservative rules of equalizing and rather apply subtle tweaks and remove obvious problems, than support personal preferences. This also means, your record will experience no significant changes from Auphonic AutoEQ if it sounds reasonably OK or pretty good already.

Conclusion

Auphonic audio post production algorithms keep getting better and better in leaps and bounds lately, offering you new Beta Features: Beta Auphonic Denoiser, Beta Auphonic Speech Recognition, and Beta Auphonic AutoEQ.
Right now we are fine-tuning all our current Beta Features with high intensity to release a new upgraded version of our Auphonic Web Service as soon as possible.
Please watch this channel for further updates – soon to come.

If you have any feedback for us or want to become a Beta user, you are very welcome to comment directly in our production interface or to contact us via email!

Phil Wilson, University of Bath will consider how Google's famous for it, Flickr's moved to Gamma, Moo are on an eternal 1.0 - yet still in institutions we plod on with a tired, slow-moving and opaque process for developing and enhancing applications. From our closed support lines to official notices on unread Web sites and applications mysteriously changing in front of a user's very eyes we look staid and tedious. But it doesn't have to be like that, we could be fast faced and interactive - but at what cost? Continuity? Uptime?

Apple released the first beta of visionOS 2.2, introducing new "Wide" and "Ultrawide" modes for the Mac Virtual Display feature on the Vision Pro headset. MacRumors reports: Apple has previously said the ultra-wide version of Mac Virtual Display is equivalent to having two physical 4K displays sitting side by side on a desk. Mac Virtual Display is now available in three sizes: Normal, Wide, and Ultrawide. visionOS 2.2 will likely be released to the public in December alongside iOS 18.2, iPadOS 18.2, macOS Sequoia 15.2, watchOS 11.2, tvOS 18.2, and other updates. Further reading: Apple Delays Cut-price Vision Headset Until 2027, Analyst Ming-Chi Kuo Says

Read more of this story at Slashdot.

AI image-generation features headline Apple's upcoming software update.

Location: Main Oversize- BQ3080.S455S45 2014

Fashion necklace and bracelet set with multi strip and tibetan spiky beads design. Solid, sturdy beads and spikes. Price: USD8.98

Fashion necklace and bracelet set with multi strip and tibetan spiky beads design.

Solid, sturdy beads and spikes.

Dimension:
Necklace - 17 inches
Bracelet - 8 inches
Extendable length - 2 inches

In this episode, Tunmi13 walks us through how to activate iOS and iPadOS beta updates.

To do this, first sign in with your Apple ID at either developer.apple.com, to access the developer beta, or beta.apple.com, to access the public beta. Then, once signed up for either program, go to Settings > General > Software update > Beta updates, and select the beta you signed up for. You can then go back one level in Settings, and the beta should appear like any other update that you can download and install.

Important: Beta versions of iOS and iPadOS may contain serious bugs, which is why it's generally advised, if possible, to install them on another device specifically designated for beta testing, or have a full and complete backup of your data in case a downgrade from the beta, which involves a full restore of your device, becomes necessary. For more information, check out this introduction to iOS and iPadOS beta testing.

La Luciérnaga se enciende para hablar de Carlos Alonso Lucio, exintegrante del M-19, quien estuvo presente junto a Ingrid Betancourt en el día en que, según relató, se encontró con el actual presidente en un profundo estado de crisis en su residencia en Bruselas, Bélgica. Lucio, ratificó la veracidad de todo lo expuesto por ella, ya que compartió ese momento con Betancourt en el encuentro con el entonces secretario de la embajada de Colombia en Bélgica. Además, la exjefe del gabinete de Gustavo Petro, Laura Sarabia, será la nueva directora del Departamento de Prosperidad Social. De acuerdo a lo que acabó de trascender, esto marcará su regreso a la rama ejecutiva. Y por último, Coviandina, empresa encargada de la concesión en la vía Bogotá- Villavicencio, anunció que este importante corredor vial tendrá un cierre temporal hasta el próximo martes 29 de agosto a las 3:00 de la mañana. 
La Luciérnaga, un espacio de humor y opinión de Caracol Radio que acompaña desde hace más de 30 años a sus oyentes en el regreso a casa.

¿Tiene relación Daily Cop con la campaña Petro Presidente?

En Caracol Radio estuvo Ingrid Betancourt, exsecuestrada por las Farc

Apple выпустила iOS 18.2 beta 3 для авторизованных разработчиков. Номер сборки — 22C5131e. Компания сообщила лишь про исправления ошибок и повышение стабильности. О другом не сообщается. Если что-то станет известно, мы обновим статью. Что нового: • вернулся старый видеоплеер в приложение Фото • начиная с iPadOS 18.2 beta 3, панель навигации в приложении TV теперь...

Apple today seeded the second public betas of upcoming iOS 18.2, iPadOS 18.2, and macOS Sequoia 15.2 updates, allowing the public to continue testing new features ahead of when the software launches. The public betas come a day after Apple provided developers with new betas.


Public beta testers can download the updates from the Settings app on each device after opting into the beta through Apple's public beta testing website. Note that Apple has also released public betas for watchOS 11.2, tvOS 18.2, and the latest HomePod software.

iOS 18.2, iPadOS 18.2, and ‌macOS Sequoia‌ introduce the next Apple Intelligence features, including the first image generation capabilities.

The update adds Image Playground, a new app for creating images based on text descriptions. You can enter anything you want, though Apple will suggest costumes, locations, items, and more to add to an image. There are options to create characters that resemble your friends and family, and you can choose a photo for ‌Image Playground‌ to use as inspiration to create a related image. Elements added to ‌Image Playground‌ creations are previewed, and there is a preview history so you can undo a change and go back to a prior version.

While ‌Image Playground‌ is a standalone app, it is also integrated into Messages, Notes, Freeform, and more. ‌Image Playground‌ does not make photorealistic images and is instead limited to animation or illustration styles.

The update also adds Genmoji, which are customizable emoji characters that you can create based on descriptions and phrases. Like ‌Image Playground‌ creations, you can base them on your friends and family, with the data pulled from the People album in Photos. You can also make characters using basic elements, and you'll get multiple ‌Genmoji‌ suggestions to choose from. You can create ‌Genmoji‌ using the emoji keyboard.

‌Genmoji‌ are limited to iOS 18.2 and iPadOS 18.2 right now, and will be coming to ‌macOS Sequoia‌ later.

Siri in iOS 18.2, iPadOS 18.2, and ‌macOS Sequoia‌ 15.2 has ChatGPT integration. If ‌Siri‌ is unable to provide an answer to a question, ‌Siri‌ will hand the request over to ChatGPT, though ‌Siri‌ will need user permission first. ChatGPT will answer the question and relay the information back through ‌Siri‌.

ChatGPT can be used to create content from scratch, including text and images. No account is required to use ChatGPT integration, and Apple and OpenAI do not store requests.

If you have an iPhone 16, there's a Visual Intelligence feature in iOS 18.2 that provides information about what's around you. Open up the camera and point it at a restaurant to get reviews, or point it at an item to search Google for it.

Some other Visual Intelligence capabilities include reading text out loud, detecting phone numbers and addresses to add them to Contacts, copying text, and summarizing text.

Apple added Writing Tools in iOS 18.1, but in iOS 18.2, you can more freely describe the tone or content change that you want to make, such as adding more action words, or turning an email into a poem.

‌Apple Intelligence‌ now supports localized English in Australia, Canada, New Zealand, South Africa, Ireland, and the UK in addition to U.S. English.

Wait List

If you've already been testing ‌Apple Intelligence‌ and are opted in, you will have access to Writing Tools, ChatGPT integration, and Visual Intelligence automatically.

There is a secondary waiting list for early access to use ‌Genmoji‌, ‌Image Playground‌, and Image Wand. You can sign up to get access in ‌Image Playground‌ or in the areas where you access ‌Genmoji‌ or Image Wand.

When you request access, you are added to a wait list for all three capabilities and you'll get a notification when the features are available for you to use. Apple will roll out access over time.

Availability and Compatibility

The public betas are available on all devices, but the ‌Apple Intelligence‌ features require a device capable of ‌Apple Intelligence‌.

Apple is still working on refining the new ‌Apple Intelligence‌ tools, and the company warns that ‌Genmoji‌, Image Wand, and ‌Image Playground‌ can sometimes give you results you weren't expecting. Apple is collecting feedback on these experiences and will refine them over time.

Release Date

Apple is expected to release the iOS 18.2, iPadOS 18.2, ‌macOS Sequoia‌ 15.2, watchOS 11.2, tvOS 18.2, and visionOS 2.2 updates in early December.
This article, "Apple Seeds Second Public Betas of iOS 18.2, iPadOS 18.2 and macOS Sequoia 15.2 With New Apple Intelligence Features" first appeared on MacRumors.com

Discuss this article in our forums

Apple today seeded the second beta of an upcoming visionOS 2.2 update to developers for testing purposes, with the beta coming a little over a week after the first visionOS 2.2 beta.


visionOS 2.2 can be installed by going to the Software Update section of the Settings app on the Vision Pro and opting into Beta Updates.

The visionOS 2.2 update adds a new feature for the Mac Virtual Display option, allowing it to be set to Wide and Ultrawide. The Ultrawide setting is equivalent to two 4K displays side-by-side, and it is a notable improvement over the current Mac Virtual Display that's available in visionOS 2.1.

The Mac Virtual Display allows the Vision Pro to be connected to a Mac with the Vision Pro serving as the display for the machine. The virtual Mac desktop works just like a standard desktop with an external display, but Vision Pro does not support multiple displays. As a solution, Apple has added the option for a wider display to maximize space.

This article, "Apple Seeds Second Beta of visionOS 2.2 to Developers With Ultrawide Mac Virtual Display" first appeared on MacRumors.com

Discuss this article in our forums

The second beta versions of iOS 18.2, MacOS Sequoia 15.2, and iPadOS 18.2 are now available for users participating in the beta testing programs for Apple system software. The new betas continue to focus on additional Apple Intelligence features, expanding beyond the writing tools, smart replies, and summary features what was initially introduced in iOS ... Read More

The third betas of iOS 18.2, iPadOS 18.2, and MacOS Sequoia 15.2, are available for beta testers of Apple system software. The latest betas continue to emphasize on new Apple Intelligence features, including ChatGPT integration, Genmoji custom Emoji creation, Image Playground for AI image generation, and more. These new AI features are in addition to ... Read More

An important mechanism of resistance to β-lactam antibiotics is via their β-lactamase–catalyzed hydrolysis. Recent work has shown that, in addition to the established hydrolysis products, the reaction of the class D nucleophilic serine β-lactamases (SBLs) with carbapenems also produces β-lactones. We report studies on the factors determining β-lactone formation by class D SBLs. We show that variations in hydrophobic residues at the active site of class D SBLs (i.e. Trp105, Val120, and Leu158, using OXA-48 numbering) impact on the relative levels of β-lactones and hydrolysis products formed. Some variants, i.e. the OXA-48 V120L and OXA-23 V128L variants, catalyze increased β-lactone formation compared with the WT enzymes. The results of kinetic and product studies reveal that variations of residues other than those directly involved in catalysis, including those arising from clinically observed mutations, can alter the reaction outcome of class D SBL catalysis. NMR studies show that some class D SBL variants catalyze formation of β-lactones from all clinically relevant carbapenems regardless of the presence or absence of a 1β-methyl substituent. Analysis of reported crystal structures for carbapenem-derived acyl-enzyme complexes reveals preferred conformations for hydrolysis and β-lactone formation. The observation of increased β-lactone formation by class D SBL variants, including the clinically observed carbapenemase OXA-48 V120L, supports the proposal that class D SBL-catalyzed rearrangement of β-lactams to β-lactones is important as a resistance mechanism.

Epoxide hydrolases (EHs) have been characterized and engineered as biocatalysts that convert epoxides to valuable chiral vicinal diol precursors of drugs and bioactive compounds. Nonetheless, the regioselectivity control of the epoxide ring opening by EHs remains challenging. Alp1U is an α/β-fold EH that exhibits poor regioselectivity in the epoxide hydrolysis of fluostatin C (compound 1) and produces a pair of stereoisomers. Herein, we established the absolute configuration of the two stereoisomeric products and determined the crystal structure of Alp1U. A Trp-186/Trp-187/Tyr-247 oxirane oxygen hole was identified in Alp1U that replaced the canonical Tyr/Tyr pair in α/β-EHs. Mutation of residues in the atypical oxirane oxygen hole of Alp1U improved the regioselectivity for epoxide hydrolysis on 1. The single site Y247F mutation led to highly regioselective (98%) attack at C-3 of 1, whereas the double mutation W187F/Y247F resulted in regioselective (94%) nucleophilic attack at C-2. Furthermore, single-crystal X-ray structures of the two regioselective Alp1U variants in complex with 1 were determined. These findings allowed insights into the reaction details of Alp1U and provided a new approach for engineering regioselective epoxide hydrolases.

G protein–coupled receptors (GPCRs) initiate signaling cascades via G-proteins and beta-arrestins (βarr). βarr-dependent actions begin with recruitment of βarr to the phosphorylated receptor tail and are followed by engagement with the receptor core. βarrs are known to act as adaptor proteins binding receptors and various effectors, but it is unclear whether in addition to the scaffolding role βarrs can allosterically activate their downstream targets. Here we demonstrate the direct allosteric activation of proto-oncogene kinase Src by GPCR–βarr complexes in vitro and establish the conformational basis of the activation. Whereas free βarr1 had no effect on Src activity, βarr1 in complex with M2 muscarinic or β2-adrenergic receptors reconstituted in lipid nanodiscs activate Src by reducing the lag phase in Src autophosphorylation. Interestingly, receptor–βarr1 complexes formed with a βarr1 mutant, in which the finger-loop, required to interact with the receptor core, has been deleted, fully retain the ability to activate Src. Similarly, βarr1 in complex with only a phosphorylated C-terminal tail of the vasopressin 2 receptor activates Src as efficiently as GPCR–βarr complexes. In contrast, βarr1 and chimeric M2 receptor with nonphosphorylated C-terminal tail failed to activate Src. Taken together, these data demonstrate that the phosphorylated GPCR tail interaction with βarr1 is necessary and sufficient to empower it to allosterically activate Src. Our findings may have implications for understanding more broadly the mechanisms of allosteric activation of downstream targets by βarrs.

Lys234 is one of the residues present in class A β-lactamases that is under selective pressure due to antibiotic use. Located adjacent to proton shuttle residue Ser130, it is suggested to play a role in proton transfer during catalysis of the antibiotics. The mechanism underpinning how substitutions in this position modulate inhibitor efficiency and substrate specificity leading to drug resistance is unclear. The K234R substitution identified in several inhibitor-resistant β-lactamase variants is associated with decreased potency of the inhibitor clavulanic acid, which is used in combination with amoxicillin to overcome β-lactamase–mediated antibiotic resistance. Here we show that for CTX-M-14 β-lactamase, whereas Lys234 is required for hydrolysis of cephalosporins such as cefotaxime, either lysine or arginine is sufficient for hydrolysis of ampicillin. Further, by determining the acylation and deacylation rates for cefotaxime hydrolysis, we show that both rates are fast, and neither is rate-limiting. The K234R substitution causes a 1500-fold decrease in the cefotaxime acylation rate but a 5-fold increase in kcat for ampicillin, suggesting that the K234R enzyme is a good penicillinase but a poor cephalosporinase due to slow acylation. Structural results suggest that the slow acylation by the K234R enzyme is due to a conformational change in Ser130, and this change also leads to decreased inhibition potency of clavulanic acid. Because other inhibitor resistance mutations also act through changes at Ser130 and such changes drastically reduce cephalosporin but not penicillin hydrolysis, we suggest that clavulanic acid paired with an oxyimino-cephalosporin rather than penicillin would impede the evolution of resistance.

Alzheimer's disease (AD) is characterized by neuronal loss and accumulation of β-amyloid-protein (Aβ) in the brain parenchyma. Sleep impairment is associated with AD and affects about 25–40% of patients in the mild-to-moderate stages of the disease. Sleep deprivation leads to increased Aβ production; however, its mechanism remains largely unknown. We hypothesized that the increase in core body temperature induced by sleep deprivation may promote Aβ production. Here, we report temperature-dependent regulation of Aβ production. We found that an increase in temperature, from 37 °C to 39 °C, significantly increased Aβ production in amyloid precursor protein-overexpressing cells. We also found that high temperature (39 °C) significantly increased the expression levels of heat shock protein 90 (Hsp90) and the C-terminal fragment of presenilin 1 (PS1-CTF) and promoted γ-secretase complex formation. Interestingly, Hsp90 was associated with the components of the premature γ-secretase complex, anterior pharynx-defective-1 (APH-1), and nicastrin (NCT) but was not associated with PS1-CTF or presenilin enhancer-2. Hsp90 knockdown abolished the increased level of Aβ production and the increased formation of the γ-secretase complex at high temperature in culture. Furthermore, with in vivo experiments, we observed increases in the levels of Hsp90, PS1-CTF, NCT, and the γ-secretase complex in the cortex of mice housed at higher room temperature (30 °C) compared with those housed at standard room temperature (23 °C). Our results suggest that high temperature regulates Aβ production by modulating γ-secretase complex formation through the binding of Hsp90 to NCT/APH-1.

Hepatocyte nuclear factor-1β (HNF-1β) is a tissue-specific transcription factor that is required for normal kidney development and renal epithelial differentiation. Mutations of HNF-1β produce congenital kidney abnormalities and inherited renal tubulopathies. Here, we show that ablation of HNF-1β in mIMCD3 renal epithelial cells results in activation of β-catenin and increased expression of lymphoid enhancer–binding factor 1 (LEF1), a downstream effector in the canonical Wnt signaling pathway. Increased expression and nuclear localization of LEF1 are also observed in cystic kidneys from Hnf1b mutant mice. Expression of dominant-negative mutant HNF-1β in mIMCD3 cells produces hyperresponsiveness to exogenous Wnt ligands, which is inhibited by siRNA-mediated knockdown of Lef1. WT HNF-1β binds to two evolutionarily conserved sites located 94 and 30 kb from the mouse Lef1 promoter. Ablation of HNF-1β decreases H3K27 trimethylation repressive marks and increases β-catenin occupancy at a site 4 kb upstream to Lef1. Mechanistically, WT HNF-1β recruits the polycomb-repressive complex 2 that catalyzes H3K27 trimethylation. Deletion of the β-catenin–binding domain of LEF1 in HNF-1β–deficient cells abolishes the increase in Lef1 transcription and decreases the expression of downstream Wnt target genes. The canonical Wnt target gene, Axin2, is also a direct transcriptional target of HNF-1β through binding to negative regulatory elements in the gene promoter. These findings demonstrate that HNF-1β regulates canonical Wnt target genes through long-range effects on histone methylation at Wnt enhancers and reveal a new mode of active transcriptional repression by HNF-1β.

Arabinogalactan proteins (AGPs) are plant proteoglycans with functions in growth and development. However, these functions are largely unexplored, mainly because of the complexity of the sugar moieties. These carbohydrate sequences are generally analyzed with the aid of glycoside hydrolases. The exo-β-1,3-galactanase is a glycoside hydrolase from the basidiomycete Phanerochaete chrysosporium (Pc1,3Gal43A), which specifically cleaves AGPs. However, its structure is not known in relation to its mechanism bypassing side chains. In this study, we solved the apo and liganded structures of Pc1,3Gal43A, which reveal a glycoside hydrolase family 43 subfamily 24 (GH43_sub24) catalytic domain together with a carbohydrate-binding module family 35 (CBM35) binding domain. GH43_sub24 is known to lack the catalytic base Asp conserved among other GH43 subfamilies. Our structure in combination with kinetic analyses reveals that the tautomerized imidic acid group of Gln263 serves as the catalytic base residue instead. Pc1,3Gal43A has three subsites that continue from the bottom of the catalytic pocket to the solvent. Subsite −1 contains a space that can accommodate the C-6 methylol of Gal, enabling the enzyme to bypass the β-1,6–linked galactan side chains of AGPs. Furthermore, the galactan-binding domain in CBM35 has a different ligand interaction mechanism from other sugar-binding CBM35s, including those that bind galactomannan. Specifically, we noted a Gly → Trp substitution, which affects pyranose stacking, and an Asp → Asn substitution in the binding pocket, which recognizes β-linked rather than α-linked Gal residues. These findings should facilitate further structural analysis of AGPs and may also be helpful in engineering designer enzymes for efficient biomass utilization.

Viral infection is one environmental factor that may contribute to the initiation of pancreatic β-cell destruction during the development of autoimmune diabetes. Picornaviruses, such as encephalomyocarditis virus (EMCV), induce a pro-inflammatory response in islets leading to local production of cytokines, such as IL-1, by resident islet leukocytes. Furthermore, IL-1 is known to stimulate β-cell expression of iNOS and production of the free radical nitric oxide. The purpose of this study was to determine whether nitric oxide contributes to the β-cell response to viral infection. We show that nitric oxide protects β-cells against virally mediated lysis by limiting EMCV replication. This protection requires low micromolar, or iNOS-derived, levels of nitric oxide. At these concentrations nitric oxide inhibits the Krebs enzyme aconitase and complex IV of the electron transport chain. Like nitric oxide, pharmacological inhibition of mitochondrial oxidative metabolism attenuates EMCV-mediated β-cell lysis by inhibiting viral replication. These findings provide novel evidence that cytokine signaling in β-cells functions to limit viral replication and subsequent β-cell lysis by attenuating mitochondrial oxidative metabolism in a nitric oxide–dependent manner.

Environmental factors, such as viral infection, are proposed to play a role in the initiation of autoimmune diabetes. In response to encephalomyocarditis virus (EMCV) infection, resident islet macrophages release the pro-inflammatory cytokine IL-1β, to levels that are sufficient to stimulate inducible nitric oxide synthase (iNOS) expression and production of micromolar levels of the free radical nitric oxide in neighboring β-cells. We have recently shown that nitric oxide inhibits EMCV replication and EMCV-mediated β-cell lysis and that this protection is associated with an inhibition of mitochondrial oxidative metabolism. Here we show that the protective actions of nitric oxide against EMCV infection are selective for β-cells and associated with the metabolic coupling of glycolysis and mitochondrial oxidation that is necessary for insulin secretion. Inhibitors of mitochondrial respiration attenuate EMCV replication in β-cells, and this inhibition is associated with a decrease in ATP levels. In mouse embryonic fibroblasts (MEFs), inhibition of mitochondrial metabolism does not modify EMCV replication or decrease ATP levels. Like most cell types, MEFs have the capacity to uncouple the glycolytic utilization of glucose from mitochondrial respiration, allowing for the maintenance of ATP levels under conditions of impaired mitochondrial respiration. It is only when MEFs are forced to use mitochondrial oxidative metabolism for ATP generation that mitochondrial inhibitors attenuate viral replication. In a β-cell selective manner, these findings indicate that nitric oxide targets the same metabolic pathways necessary for glucose stimulated insulin secretion for protection from viral lysis.

The Wnt/β-catenin pathway is one of the major pathways that regulates embryonic development, adult homeostasis, and stem cell self-renewal. In this pathway, transcription factors T-cell factor and lymphoid enhancer factor (TCF/LEF) serve as a key switch to repress or activate Wnt target gene transcription by recruiting repressor molecules or interacting with the β-catenin effector, respectively. It has become evident that the protein stability of the TCF/LEF family members may play a critical role in controlling the activity of the Wnt/β-catenin signaling pathway. However, factors that regulate the stability of TCF/LEFs remain largely unknown. Here, we report that pVHL binding protein 1 (VBP1) regulates the Wnt/β-catenin signaling pathway by controlling the stability of TCF/LEFs. Surprisingly, we found that either overexpression or knockdown of VBP1 decreased Wnt/β-catenin signaling activity in both cultured cells and zebrafish embryos. Mechanistically, VBP1 directly binds to all four TCF/LEF family members and von Hippel-Lindau tumor-suppressor protein (pVHL). Either overexpression or knockdown of VBP1 increases the association between TCF/LEFs and pVHL and then decreases the protein levels of TCF/LEFs via proteasomal degradation. Together, our results provide mechanistic insights into the roles of VBP1 in controlling TCF/LEFs protein stability and regulating Wnt/β-catenin signaling pathway activity.

Elevated levels of fasting insulin release and insufficient glucose-stimulated insulin secretion (GSIS) are hallmarks of diabetes. Studies have established cross-talk between integrin signaling and insulin activity, but more details of how integrin-dependent signaling impacts the pathophysiology of diabetes are needed. Here, we dissected integrin-dependent signaling pathways involved in the regulation of insulin secretion in β-cells and studied their link to the still debated autocrine regulation of insulin secretion by insulin/insulin-like growth factor (IGF) 2–AKT signaling. We observed for the first time a cooperation between different AKT isoforms and focal adhesion kinase (FAK)–dependent adhesion signaling, which either controlled GSIS or prevented insulin secretion under fasting conditions. Indeed, β-cells form integrin-containing adhesions, which provide anchorage to the pancreatic extracellular matrix and are the origin of intracellular signaling via FAK and paxillin. Under low-glucose conditions, β-cells adopt a starved adhesion phenotype consisting of actin stress fibers and large peripheral focal adhesion. In contrast, glucose stimulation induces cell spreading, actin remodeling, and point-like adhesions that contain phospho-FAK and phosphopaxillin, located in small protrusions. Rat primary β-cells and mouse insulinomas showed an adhesion remodeling during GSIS resulting from autocrine insulin/IGF2 and AKT1 signaling. However, under starving conditions, the maintenance of stress fibers and the large adhesion phenotype required autocrine IGF2-IGF1 receptor signaling mediated by AKT2 and elevated FAK-kinase activity and ROCK-RhoA levels but low levels of paxillin phosphorylation. This starved adhesion phenotype prevented excessive insulin granule release to maintain low insulin secretion during fasting. Thus, deregulation of the IGF2 and adhesion-mediated signaling may explain dysfunctions observed in diabetes.

Transient receptor potential vanilloid 1 (TRPV1) channel is a multimodal receptor that is responsible for nociceptive, thermal, and mechanical sensations. However, which biomolecular partners specifically interact with TRPV1 remains to be elucidated. Here, we used cDNA library screening of genes from mouse dorsal root ganglia combined with patch-clamp electrophysiology to identify the voltage-gated potassium channel auxiliary subunit Kvβ1 physically interacting with TRPV1 channel and regulating its function. The interaction was validated in situ using endogenous dorsal root ganglia neurons, as well as a recombinant expression model in HEK 293T cells. The presence of Kvβ1 enhanced the expression stability of TRPV1 channels on the plasma membrane and the nociceptive current density. Surprisingly, Kvβ1 interaction also shifted the temperature threshold for TRPV1 thermal activation. Using site-specific mapping, we further revealed that Kvβ1 interacted with the membrane-distal domain and membrane-proximal domain of TRPV1 to regulate its membrane expression and temperature-activation threshold, respectively. Our data therefore suggest that Kvβ1 is a key element in the TRPV1 signaling complex and exerts dual regulatory effects in a site-specific manner.

AMP-activated protein kinase (AMPK) is a key regulator of energy metabolism that phosphorylates a wide range of proteins to maintain cellular homeostasis. AMPK consists of three subunits: α, β, and γ. AMPKα and β are encoded by two genes, the γ subunit by three genes, all of which are expressed in a tissue-specific manner. It is not fully understood, whether individual isoforms have different functions. Using RNA-Seq technology, we provide evidence that the loss of AMPKβ1 and AMPKβ2 lead to different gene expression profiles in human induced pluripotent stem cells (hiPSCs), indicating isoform-specific function. The knockout of AMPKβ2 was associated with a higher number of differentially regulated genes than the deletion of AMPKβ1, suggesting that AMPKβ2 has a more comprehensive impact on the transcriptome. Bioinformatics analysis identified cell differentiation as one biological function being specifically associated with AMPKβ2. Correspondingly, the two isoforms differentially affected lineage decision toward a cardiac cell fate. Although the lack of PRKAB1 impacted differentiation into cardiomyocytes only at late stages of cardiac maturation, the availability of PRKAB2 was indispensable for mesoderm specification as shown by gene expression analysis and histochemical staining for cardiac lineage markers such as cTnT, GATA4, and NKX2.5. Ultimately, the lack of AMPKβ1 impairs, whereas deficiency of AMPKβ2 abrogates differentiation into cardiomyocytes. Finally, we demonstrate that AMPK affects cellular physiology by engaging in the regulation of hiPSC transcription in an isoform-specific manner, providing the basis for further investigations elucidating the role of dedicated AMPK subunits in the modulation of gene expression.

Daphne E.C. Boer
Dec 23, 2020; 0:jlr.RA120001043v1-jlr.RA120001043
Research Articles

Deficiency of glucocerebrosidase (GBA), a lysosomal β-glucosidase, causes Gaucher disease. The enzyme hydrolyzes β-glucosidic substrates and transglucosylates cholesterol to cholesterol-β-glucoside. Here we show that recombinant human GBA also cleaves β-xylosides and transxylosylates cholesterol. The xylosyl-cholesterol formed acts as acceptor for subsequent formation of di-xylosyl-cholesterol. Common mutant forms of GBA from patients with Gaucher disease with reduced β-glucosidase activity were similarly impaired in β-xylosidase, transglucosidase and transxylosidase activities, except for a slightly reduced xylosidase/glucosidase activity ratio of N370S GBA and a slightly reduced transglucosylation/glucosidase activity ratio of D409H GBA. XylChol was found to be reduced in spleen from Gaucher disease patients. The origin of newly identified XylChol in mouse and human tissues was investigated. Cultured human cells exposed to exogenous β-xylosides generated XylChol in a manner dependent on active lysosomal GBA but not the cytosol-facing β-glucosidase GBA2. We later sought an endogenous β-xyloside acting as donor in transxylosylation reactions, identifying xylosylated ceramide (XylCer) in cells and tissues that serve as donor in the formation of XylChol. UDP-glucosylceramide synthase (GCS) was unable to synthesize XylChol but could catalyse formation of XylCer. Thus, food-derived β-D-xyloside and XylCer are potential donors for the GBA-mediated formation of XylChol in cells. The enzyme GCS produces XylCer at a low rate. Our findings point to further catalytic versatility of GBA and prompt a systematic exploration of the distribution and role of xylosylated lipids.

The acyltransferase LCAT mediates FA esterification of plasma cholesterol. In vitro studies have shown that LCAT also FA-esterifies several oxysterols, but in vivo evidence is lacking. Here, we measured both free and FA-esterified forms of sterols in 206 healthy volunteers and 8 individuals with genetic LCAT deficiency, including familial LCAT deficiency (FLD) and fish-eye disease (FED). In the healthy volunteers, the mean values of the ester-to-total molar ratios of the following sterols varied: 4β-hydroxycholesterol (4βHC), 0.38; 5,6α-epoxycholesterol (5,6αEC), 0.46; 5,6β-epoxycholesterol (5,6βEC), 0.51; cholesterol, 0.70; cholestane-3β,5α,6β-triol (CT), 0.70; 7-ketocholesterol (7KC), 0.75; 24S-hydroxycholesterol (24SHC), 0.80; 25-hydroxycholesterol (25HC), 0.81; 27-hydroxycholesterol (27HC), 0.86; and 7α-hydroxycholesterol (7αHC), 0.89. In the individuals with LCAT deficiency, the plasma levels of the FA-esterified forms of cholesterol, 5,6αEC, 5,6βEC, CT, 7αHC, 7KC, 24SHC, 25HC, and 27HC, were significantly lower than those in the healthy volunteers. The individuals with FLD had significantly lower FA-esterified forms of 7αHC, 24SHC, and 27HC than those with FED. It is of note that, even in the three FLD individuals with negligible plasma cholesteryl ester, substantial amounts of the FA-esterified forms of 4βHC, 5,6αEC, 7αHC, 7KC, and 27HC were present. We conclude that LCAT has a major role in the FA esterification of many plasma oxysterols but contributes little to the FA esterification of 4βHC. Substantial FA esterification of 4βHC, 5,6αEC, 7αHC, 7KC, and 27HC is independent of LCAT.

Atherosclerosis is characterized by the pathological accumulation of cholesterol-laden macrophages in the arterial wall. Atherosclerosis is also the main underlying cause of CVDs, and its development is largely driven by elevated plasma cholesterol. Strong epidemiological data find an inverse association between plasma β-carotene with atherosclerosis, and we recently showed that β-carotene oxygenase 1 (BCO1) activity, responsible for β-carotene cleavage to vitamin A, is associated with reduced plasma cholesterol in humans and mice. In this study, we explore whether intact β-carotene or vitamin A affects atherosclerosis progression in the atheroprone LDLR-deficient mice. Compared with control-fed Ldlr^–/– mice, β-carotene-supplemented mice showed reduced atherosclerotic lesion size at the level of the aortic root and reduced plasma cholesterol levels. These changes were absent in Ldlr^–/–/Bco1^–/– mice despite accumulating β-carotene in plasma and atherosclerotic lesions. We discarded the implication of myeloid BCO1 in the development of atherosclerosis by performing bone marrow transplant experiments. Lipid production assays found that retinoic acid, the active form of vitamin A, reduced the secretion of newly synthetized triglyceride and cholesteryl ester in cell culture and mice. Overall, our findings provide insights into the role of BCO1 activity and vitamin A in atherosclerosis progression through the regulation of hepatic lipid metabolism.

The adipocyte-derived hormone leptin increases trafficking of KATP and Kv2.1 channels to the pancreatic β-cell surface, resulting in membrane hyperpolarization and suppression of insulin secretion. We have previously shown that this effect of leptin is mediated by the NMDA subtype of glutamate receptors (NMDARs). It does so by potentiating NMDAR activity, thus enhancing Ca2+ influx and the ensuing downstream signaling events that drive channel trafficking to the cell surface. However, the molecular mechanism by which leptin potentiates NMDARs in β-cells remains unknown. Here, we report that leptin augments NMDAR function via Src kinase–mediated phosphorylation of the GluN2A subunit. Leptin-induced membrane hyperpolarization diminished upon pharmacological inhibition of GluN2A but not GluN2B, indicating involvement of GluN2A-containing NMDARs. GluN2A harbors tyrosine residues that, when phosphorylated by Src family kinases, potentiate NMDAR activity. We found that leptin increases phosphorylation of Tyr-418 in Src, an indicator of kinase activation. Pharmacological inhibition of Src or overexpression of a kinase-dead Src mutant prevented the effect of leptin, whereas a Src kinase activator peptide mimicked it. Using mutant GluN2A overexpression, we show that Tyr-1292 and Tyr-1387 but not Tyr-1325 are responsible for the effect of leptin. Importantly, β-cells from db/db mice, a type 2 diabetes mouse model lacking functional leptin receptors, or from obese diabetic human donors failed to respond to leptin but hyperpolarized in response to NMDA. Our study reveals a signaling pathway wherein leptin modulates NMDARs via Src to regulate β-cell excitability and suggests NMDARs as a potential target to overcome leptin resistance.

Holly Oakley
Oct 4, 2006; 26:10129-10140
Neurobiology of Disease

Donna M. Wilcock
May 1, 2003; 23:3745-3751
Development Plasticity Repair

Robbert Havekes
Dec 12, 2012; 32:18137-18149
BehavioralSystemsCognitive

Holly Oakley
Oct 4, 2006; 26:10129-10140
Neurobiology of Disease