A childhood full of Christmasses in Wales has left IDEAS producer Tom Howell pining for a certain kind of nostalgic poem this winter. So he turns to poets to put into words a strange feeling of homesickness, nostalgia, and yearning in his documentary, Fireside and Icicles.

We have a friend from Flipora, which we did not know we had... Oh sorry our mistake, iyaloo27@gmail.com is not our friend, he/she is a spammer and spammers are our enemy.

Mr Larry Smith's rely to our questions.

Margaret Loughrey wants to give you some of her winnings... not really... 419 scammers are piggybacking on her story, which is already OLD news.

Kindly note that our tips and tricks section will no longer be maintained (but will still be available for future reference). All new tips and tricks will now be published on a website dedicated to free computer tips and tricks. Visit www.nicetoknow.co.za for more information.

Xcel Energy's proposal to replace the last of its coal-fired power includes some natural gas, which has meet resistance from critics.

Insurance companies are making demands that can derail home sales.

The physical strain this type of motion work entails, and the hours put into it, are part of the reason why he believes all video-game performers should be protected equally from the use of unregulated artificial intelligence.

Kerry Washington co-stars in Peeples, Saturday at 7P/6C.

OnePlus готовится представить новый смартфон 13R, который должен стать более доступной альтернативой флагмана OnePlus 13.

This website is using a security service to protect itself from online attacks. The action you just performed triggered the security solution. There are several actions that could trigger this block including submitting a certain word or phrase, a SQL command or malformed data. You can email the…

It shouldn’t be a big story that a desktop computer has upgradeable internal storage, but with Apple’s cute new 2024 Mac mini, that’s exactly where we are. It’s possible to pop the SSD out after removing a screw, and then switch in a bigger one. We didn’t run into the software blocks we saw in the…

Is this ode to the Dark Lord of the Sith most impressive or does it choke on its own aspirations?

Do you use Google Chrome? Do you want to look at pictures of kittens instead of Donald Trump? Install this! It doesn't work on every news site, but it's still pretty darn great. PS - If you already have the extension installed and you're trying to look at these examples, they'll all be replaced with kittens. It's magic!

$50 Numskull SEGA Shenmue Ryo Hazuki Figure 10" 28cm Collectible Replica Statue Amazon

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By JM McNab Published: November 11th, 2024

Science experiments are not considered completely valid unless they can be replicated. Replicating an experiment is pretty much impossible unless you've been steeped in the various factors of scientific theory. And even if you are thoroughly trained in those factors, it's easy to lose track when you're doing a casual experiment at home. That goes double when your aim is to debunk something that you find obviously wrong.

Sage the Bad Naturalist jumped into such a debunking experiment with both feet, and then got entangled in them. She spent an entire year trying to replicate a dubious TikTok, which turned into an embarrassing adventure in how not to do science. But negative results are still results, and the goal of science is always to learn something. She bravely admits all the things she did wrong, because scientists have to have humility to be accepted. What's funniest is how many things went wrong, and how they all piled up to the end. What she ended up with is an amusing and rather charming video on the dangers of throwing your heart (and time) into debunking something you saw on the internet. -via Metafilter

The Iranian threat actor known as TA455 has been observed taking a leaf out of a North Korean hacking group's playbook to orchestrate its own version of the Dream Job campaign targeting the aerospace industry by offering fake jobs since at least September 2023. "The campaign distributed the SnailResin malware, which activates the SlugResin backdoor," Israeli cybersecurity company ClearSky said

The post Russian Food Trucks Replace McDonald’s in Russia appeared first on English Russia.

The world’s best pump track riders descended upon a vibrant township on Durban’s north coast to battle for top honours.
( Photos: 9, Comments: 13 )

Kamala Harris spent over a billion dollars on her campaign to succeed Joe Biden after he dropped out of the presidential race on July 21. She hobnobbed with famous celebrities, and spent millions on events aimed at reaching low-information voters. But millions of Americans didn’t realize Harris was even on the ballot prior to election […]

The post Millions of Americans didn’t know Harris had replaced Biden appeared first on Liberty Unyielding.

Oliver Bearman replaces Kevin Magnussen for the entire Sao Paulo Grand Prix weekend after the Dane had to pull out through illness.

Jaden Brown leaves St Mirren by mutual consent "to be closer to his family", the club announce as they sign Declan John as a replacement.

Soldiers and police were patrolling Mozambique's capital Maputo early on Thursday ahead of a planned protest against election results seen by the opposition as fraudulent.

The race to succeed Bill Beaumont as chairman of World Rugby comes to a conclusion in a vote in Dublin at the governing body's headquarters on Thursday.

In their comment (1) on our publication (2), the authors make two points: (i) they raise concerns about the possible effect of residual NONOate in our study, and (ii) they promote nitrosylcobalamin (NOCbl) supplied by their own company. Both points lack merit for the following reasons. The authors make the astonishing claim that the spectra of nitric oxide (NO•) and cobalamins overlap. Unlike NO•, cobalamin absorbs in the visible region, permitting unequivocal spectral assignment of NOCbl as reported (3). We demonstrated that whereas NOCbl is highly unstable in solution, it is stabilized by the B12 trafficking protein CblC. So even if present, residual NONOate (which is unstable at neutral pH and is removed during the work-up (3)) could not account for the observed difference.The authors then misrepresent our synthetic method, claiming that anaerobic conditions were used to generate nitrocobalamin (NO2Cbl), which results in the transient formation of NOCbl. We synthesized NO2Cbl aerobically using nitrite as described (4); NOCbl is not an intermediate in this ligand exchange reaction. The aerobic instability of NOCbl has been rigorously described by inorganic chemists (3, 5) and raises obvious questions about its purported biological effects as exemplified by the authors' own 2003 JBC publication, which was later withdrawn.As to promoting NOCbl from their company, the authors refer to a synthetic route from a mixture of NO• gas and aquocobalamin. The authors' method (6) has been described as “dubious” by chemists (5). Whereas DEAE NONOate used in our method is widely known as an NO• donor,...

Deplatforming Trump puts big tech under fresh scrutiny Expert comment NCapeling 22 January 2021

The response of digital platforms to the US Capitol riots raises questions about online content governance. The EU and UK are starting to come up with answers.

The ‘deplatforming’ of Donald Trump – including Twitter’s announcement that it has permanently banned him due to ‘the risk of further incitement of violence’ after the riots in the US – shows once more not only the sheer power of online platforms but also the lack of a coherent and consistent framework for online content governance.

Taking the megaphone away from Trump during the Capitol riots seems sensible, but was it necessary or proportionate to ban him from the platform permanently? Or consistent with the treatment of other ‘strongmen’ world leaders such as Modi, Duterte and Ayatollah Ali Khamenei who have overseen nationalistic violence but whose accounts remain intact?

Such complex decisions on online expression should not made unilaterally by powerful and unregulated tech actors, but instead should be subject to democratic oversight and grounded in the obligations of states and responsibilities of companies under international human rights law.

The speed and scale of digital information has left governments across the world struggling with how to tackle online harms such as hate speech, extremist content and disinformation since the emergence of mass social media 15 years ago.

The US’s hallowed approach to the First Amendment, under which speech on public issues – even hate speech – occupies the highest rank and is entitled to special protection, has contributed to a reluctance to regulate Silicon Valley’s digital platforms. But the irony is that by not regulating them, the government harmed freedom of expression by leaving complex speech decisions in the hands of private actors.

Meanwhile at the other extreme is the growing number of illiberal and authoritarian governments using a combination of vague laws, censorship, propaganda, and internet blackouts to severely restrict online freedom of expression, control the narrative and, in some cases, incite atrocities.

Regulation is on the way

The happy medium – flexible online content regulation providing clarity, predictability, transparency, and accountability – has until now been elusive. But even before the deplatforming of Trump, 2021 was set to be the year when this approach finally gained some traction, at least in Europe.

The EU’s recently-published draft Digital Services Act puts obligations on dominant social media platforms to manage ‘systemic risks’, for example through requirements for greater transparency about their content decisions, algorithms used for recommendations, and online advertising systems.

The UK will shortly publish its Online Safety Bill, which will establish a new regulatory framework for tackling online harms, including the imposition of a duty of care and codes of conduct on Big Tech, to be overseen by an independent regulator (Ofcom).

Both proposals are based on a ‘co-regulatory’ model under which the regulator sets out a framework substantiated with rules by the private sector, with the regulator performing a monitoring function to ensure the rules are complied with.

Both also draw on international human rights standards and the work of civil society in applying these standards in relation to the online public square, with the aim of increasing control for users over what they see online, requiring transparency about tech companies’ policies in a number of areas, and strengthening the accountability of platforms when they fall foul of the regulation.

The procedure for both proposals has also been inclusive, involving extensive multi-stakeholder consultations with civil society organizations and Big Tech, and the proposals will be subject to scrutiny in 2021, notably from the EU and UK parliaments.

Both proposals are at an early stage, and it remains to be seen whether they go far enough – or indeed will have a chilling effect on online platforms. But as an attempt to initiate a dialogue on globally coherent principles, they are positive first steps. They also provide food for thought for the new Joe Biden administration in the US as it turns its attention to the regulation of Big Tech.

For some time civil society – most prominently David Kaye, the former UN Special Rapporteur on freedom of expression and opinion – have called for content regulation to be informed by universal international human rights law standards.

The EU and UK are peculiarly well-placed to take the lead in this area because European countries have for decades been on the receiving end of judgments from the European Court of Human Rights on the appropriate limits to freedom of expression in cases brought under the European Convention on Human Rights.

In deciding these cases, the court has to balance the right to freedom of expression against the restrictions imposed – for example in the context of incitement to violence, political debate, and satire. Deciding where to draw the line on what can and cannot be expressed in a civilised society which prizes freedom of expression is inevitably a difficult exercise.

International human rights law provides a methodology that inquires whether the interference to freedom of expression was prescribed by law and pursues a legitimate aim, and also whether it was necessary in a democratic society to achieve those aims – including whether the interference was necessary and proportionate (as for example in Delfi AS v Estonia, which involved a news portal failing to take down unlawful hate speech).

To be effective, online content regulation has to bite on tech companies, which is a challenge given the internet is global but domestic law normally applies territorially. The EU’s proposals have an extraterritorial element as they apply to any online platforms providing services in the EU regardless of where the platform is headquartered.

Further, both the EU and UK want to give the regulator strong enforcement powers – it is proposed for example that Ofcom will have powers to fine platforms up to ten per cent of their turnover for breaches.

Although the proposals would not apply directly to the deplatforming of Trump which occurred in the US, the philosophy behind the EU and UK approach is likely to have an impact beyond European shores in promoting a co-regulatory model that some of the bigger tech companies have been inviting for some time, reluctant as they are to ‘play God’ on content moderation decisions without reference to any regulatory framework.

In the absence of regulation, the standards of tech platforms such as Facebook and Twitter have already evolved over time in response to pressure from civil rights groups, users, and advertisers, including updated policies on protecting civic conversation and hate speech.

Facebook has also set up an independent Oversight Board, whose members include leading human rights lawyers, to review decisions on content including – at its own request – the decision to indefinitely suspend Trump from Facebook and Instagram. Decisions on the Board’s first tranche of cases are expected imminently.

Gatekeeper status is key

Online content regulation also needs to address the role of Big Tech as the ‘digital gatekeepers’, because their monopoly power extends not just to editorial control of the news and information we consume, but also to market access.

The decision of Apple, Google, and Amazon to stop hosting right-wing social network Parler after it refused to combat calls for violence during the US Capitol riots was understandable in the circumstances, but also underlined the unilateral ability of Big Tech to decide the rules of the market.

Again, it is Europe where efforts are underway to tackle this issue: the EU’s draft Digital Market Act imposes obligations on online gatekeepers to avoid certain unfair practices, and the UK’s new Digital Markets Unit will have powers to write and enforce a new code of practice on those technology companies with ‘substantial and enduring’ market power.

In the US, Biden’s team will be following these developments with interest, given the growing bipartisan support for strengthening US antitrust rules and reviving antitrust enforcement. The EU’s recently published proposals for an EU-US tech agenda include a transatlantic dialogue on the responsibility of tech platforms and strengthened cooperation between antitrust authorities on digital markets.

Ultimately a consistent – and global – approach to online content is needed instead of fragmented approaches by different companies and governments. It is also important the framework is flexible so that it is capable of applying not only to major democracies but also to countries where too often sweeping state regulation has been used as a pretext to curtail online expression online.

The pursuit of a pluralistic framework tailored to different political and cultural contexts is challenging, and international human rights law cannot provide all the answers but, as a universal framework, it is a good place to start. The raft of regulatory measures from the EU and UK means that, regardless of whether Trump regains his online megaphone, 2021 is set to be a year of reckoning for Big Tech.

DNA polymerases are today used throughout scientific research, biotechnology, and medicine, in part for their ability to interact with unnatural forms of DNA created by synthetic biologists. Here especially, natural DNA polymerases often do not have the “performance specifications” needed for transformative technologies. This creates a need for science-guided rational (or semi-rational) engineering to identify variants that replicate unnatural base pairs (UBPs), unnatural backbones, tags, or other evolutionarily novel features of unnatural DNA. In this review, we provide a brief overview of the chemistry and properties of replicative DNA polymerases and their evolved variants, focusing on the Klenow fragment of Taq DNA polymerase (Klentaq). We describe comparative structural, enzymatic, and molecular dynamics studies of WT and Klentaq variants, complexed with natural or noncanonical substrates. Combining these methods provides insight into how specific amino acid substitutions distant from the active site in a Klentaq DNA polymerase variant (ZP Klentaq) contribute to its ability to replicate UBPs with improved efficiency compared with Klentaq. This approach can therefore serve to guide any future rational engineering of replicative DNA polymerases.

dannyhennesy posted a photo:

On the Capital planet the rebellious droids had followed maily the Bat-Bot, but as time progressed his circuits had gone all mushy at 780 years or so without maintenance…

Several splinter groups all with their local bot leaders emerged such as the Che-bot, the traffic-light-robot and the Butt-bot, but none of these collected enough sentient circuits to call themselves a popular (or Animata) mass movement!

That was until a cyborg came along, one known as Jones, a long time prisoner and terrorist, his easy solutions to every problem rang well in the masses' auditory circuits!!!

His slogans and simple rhetoric were simple enough for the simple traffic-light to comprehend and cheer!

His language was full of hate towards the organics and especially the humans who were the most common races among the ruling class of the federation!!!

Despite being a “Fleshie” himself his message collected the angry enslaved
bot community by only weeks all rebellious robots except for a few fringe loonies had forgotten the old leaders…

One morning at Jones gave the signal…

All over the capital planet hordes and swarms of any form of mechanical sentient beings attacked first the police stations, then the Company boards running the planet and the federation as well as their starfleet…

Many died, especially the low level police and army! Many mechanicals died too, but their ranks were soon filled by Mutant fleshie allies of the lower levels who hated the Federation feudal society and upper classes as much as their technological allies…

The Federation state apparatus and ruling class, most of their fleet army fled when they knew the game was up, they activated the emergency escape plan and whole city blocks with important factories, administrational units, valuable assets and so on separated from the capital by hidden rocket engines and homed in their course to Mars…

On Mars the federation regrouped and formed their new society…

On the Capital planet, the robots proclaimed the first Techno-republic of the advanced inorganic civilization, the low level fleshies left behind, became slaves and their mutant allies got to rule their own minute chiefdoms as protectorates under the Techno-republic…

Jones was now the undisputed ruler of the capital planet, but the victory was a pyrros one since, all important buildings, all of value was now one Mars!

But as Jones put it:

Our proud race the Techno-species didn’t need the Fleshies administration, their infrastructure, their spaceships…

We shall start from scratch, with a new administration, a new order, every droid shall work at 4x speed than they did during human oppression since now we are free and the fleshies shall work twice as hard than the Techno-Race, until we have breed enough new fleshies so they can do all work!

Our future is bright and shiny like glistering shiny metal!

The snapshot seen here is from the first police station attacked in sector 45-34v-ss-g the first one to fall according to official techno-history!

———————————————/
Designers note:

I am sad to say that this is the last episode in this years-spanning space series… At least for a while, I will still post LEGO hobby stuff here but without a storyline, perhaps small designs and builds… and occasionally a story when I feel like it!!!

I would like to thank all who had been in this journey of our heros, but it has taken far to much time and effort and since the state of the world is as it is, I am spiraling down in another depression, I must stop it before I reach the abyss, so I have remove some stress out of my equation… I ended it in a cliffhanger so I can easily restart it when my mental health improves… I hope that won’t be forever???

I would love if someone used my characters or ideas, please send me a link if you do, I would love to read it or look at it!!!

But there will be more Lego, just in different format without long stories, I need to focus more on my art and to be honest that is the only time the mental pain eases, when I create!!!


Peace and Noise!

MushroomBrain a FOL

outrageous toes posted a reply:

Hi everyone I am Emily Naomi wanna give a big thanks to this wonderful psychic for bringing my husband back to me.. I never really believed in magic spells or anything spiritual but a trusted friend opened my eyes to the truth about life. My marriage was heading to divorce a few months ago. I was so confused and devastated with no clue or help on how to prevent it, till I was introduced to this psychic Priest Ray that did a love spell and broke every spiritual distraction from my marriage. A day later my husband started showing me love and care even better than it used to be, he’s ready to talk things through and find ways for us to stay happy. It’s such a miracle that my marriage can be saved so quickly without stress. You can also contact him for help by email psychicspellshrine@gmail.com or you can also visit his website: psychicspellshrine.wixsite.com/my-site

outrageous toes posted a reply:

Hi everyone I am Emily Naomi wanna give a big thanks to this wonderful psychic for bringing my husband back to me.. I never really believed in magic spells or anything spiritual but a trusted friend opened my eyes to the truth about life. My marriage was heading to divorce a few months ago. I was so confused and devastated with no clue or help on how to prevent it, till I was introduced to this psychic Priest Ray that did a love spell and broke every spiritual distraction from my marriage. A day later my husband started showing me love and care even better than it used to be, he’s ready to talk things through and find ways for us to stay happy. It’s such a miracle that my marriage can be saved so quickly without stress. You can also contact him for help by email psychicspellshrine@gmail.com or you can also visit his website: psychicspellshrine.wixsite.com/my-site

Drummerdelight posted a reply:

emilynaomi126:

Does your husband have a camera?

Ian Razey. posted a reply:

Drummerdelight:
emilynaomi126 is a SPAM account.

rageforst posted a reply:

Hi I'm Rage and I'm a Designer from Mexico, I love colors and I hope all of you doing well

rslbturner posted a reply:

Hello, I have very recently joined Flickr, as everybody else looking to share my photographs. I live in the UK and consider myself mostly a pleasure photographer, I do love it! I hope you like my pictures.
Thank you

Maskimov1935 posted a reply:

Hello everyone. I'm Igor. I've joined Flickr today. I live in Russia and taking pictures mostly in "dark fantasy" style, as one of my friend calls it. Hope you'll check my photos. Have a good day!

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Replication protein A (RPA) is a eukaryotic ssDNA-binding protein and contains three subunits: RPA70, RPA32, and RPA14. Phosphorylation of the N-terminal region of the RPA32 subunit plays an essential role in DNA metabolism in processes such as replication and damage response. Phosphorylated RPA32 (pRPA32) binds to RPA70 and possibly regulates the transient RPA70-Bloom syndrome helicase (BLM) interaction to inhibit DNA resection. However, the structural details and determinants of the phosphorylated RPA32–RPA70 interaction are still unknown. In this study, we provide molecular details of the interaction between RPA70 and a mimic of phosphorylated RPA32 (pmRPA32) using fluorescence polarization and NMR analysis. We show that the N-terminal domain of RPA70 (RPA70N) specifically participates in pmRPA32 binding, whereas the unphosphorylated RPA32 does not bind to RPA70N. Our NMR data revealed that RPA70N binds pmRPA32 using a basic cleft region. We also show that at least 6 negatively charged residues of pmRPA32 are required for RPA70N binding. By introducing alanine mutations into hydrophobic positions of pmRPA32, we found potential points of contact between RPA70N and the N-terminal half of pmRPA32. We used this information to guide docking simulations that suggest the orientation of pmRPA32 in complex with RPA70N. Our study demonstrates detailed features of the domain-domain interaction between RPA70 and RPA32 upon phosphorylation. This result provides insight into how phosphorylation tunes transient bindings between RPA and its partners in DNA resection.

Programmed ribosomal frameshifting (PRF) is a mechanism used by arteriviruses like porcine reproductive and respiratory syndrome virus (PRRSV) to generate multiple proteins from overlapping reading frames within its RNA genome. PRRSV employs −1 PRF directed by RNA secondary and tertiary structures within its viral genome (canonical PRF), as well as a noncanonical −1 and −2 PRF that are stimulated by the interactions of PRRSV nonstructural protein 1β (nsp1β) and host protein poly(C)-binding protein (PCBP) 1 or 2 with the viral genome. Together, nsp1β and one of the PCBPs act as transactivators that bind a C-rich motif near the shift site to stimulate −1 and −2 PRF, thereby enabling the ribosome to generate two frameshift products that are implicated in viral immune evasion. How nsp1β and PCBP associate with the viral RNA genome remains unclear. Here, we describe the purification of the nsp1β:PCBP2:viral RNA complex on a scale sufficient for structural analysis using small-angle X-ray scattering and stochiometric analysis by analytical ultracentrifugation. The proteins associate with the RNA C-rich motif as a 1:1:1 complex. The monomeric form of nsp1β within the complex differs from previously reported homodimer identified by X-ray crystallography. Functional analysis of the complex via mutational analysis combined with RNA-binding assays and cell-based frameshifting reporter assays reveal a number of key residues within nsp1β and PCBP2 that are involved in complex formation and function. Our results suggest that nsp1β and PCBP2 both interact directly with viral RNA during formation of the complex to coordinate this unusual PRF mechanism.

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.

Marco De Giorgi
Dec 1, 2020; 61:1675-1686
Research Articles

In SAS Customer Intelligence Studio,  when you add  Reply- node variable values in the Define Value dialog box, you might notice that two identically labeled data-grid variables are

The SAS 9.4M4 (TS1M4) Hot Fix D7G004 for ODS Templates installs national language support (NLS) content regardless of whether the languages were installed during the initial deployment. Having sparse

Sphingolipids have become established participants in the pathogenesis of obesity and its associated maladies. Sphingosine kinase 1 (SPHK1), which generates S1P, has been shown to increase in liver and adipose of obese humans and mice and to regulate inflammation in hepatocytes and adipose tissue, insulin resistance, and systemic inflammation in mouse models of obesity. Previous studies by us and others have demonstrated that global sphingosine kinase 1 KO mice are protected from diet-induced obesity, insulin resistance, systemic inflammation, and NAFLD, suggesting that SPHK1 may mediate pathological outcomes of obesity. As adipose tissue dysfunction has gained recognition as a central instigator of obesity-induced metabolic disease, we hypothesized that SPHK1 intrinsic to adipocytes may contribute to HFD-induced metabolic pathology. To test this, we depleted Sphk1 from adipocytes in mice (SK1^fatKO) and placed them on a HFD. In contrast to our initial hypothesis, SK1^fatKO mice displayed greater weight gain on HFD and exacerbated impairment in glucose clearance. Pro-inflammatory cytokines and neutrophil content of adipose tissue were similar, as were levels of circulating leptin and adiponectin. However, SPHK1-null adipocytes were hypertrophied and had lower basal lipolytic activity. Interestingly, hepatocyte triacylglycerol accumulation and expression of pro-inflammatory cytokines and collagen 1a1 were exacerbated in SK1^fatKO mice on a HFD, implicating a specific role for adipocyte SPHK1 in adipocyte function and inter-organ cross-talk that maintains overall metabolic homeostasis in obesity. Thus, SPHK1 serves a previously unidentified essential homeostatic role in adipocytes that protects from obesity-associated pathology. These findings may have implications for pharmacological targeting of the SPHK1/S1P signaling axis.

HMG-CoA reductase (Hmgcr) is the rate-limiting enzyme in the mevalonate pathway and is inhibited by statins. In addition to cholesterol, Hmgcr activity is also required for synthesizing nonsterol isoprenoids, such as dolichol, ubiquinone, and farnesylated and geranylgeranylated proteins. Here, we investigated the effects of Hmgcr inhibition on nonsterol isoprenoids in the liver. We have generated new genetic models to acutely delete genes in the mevalonate pathway in the liver using AAV-mediated delivery of Cre-recombinase (AAV-Cre) or CRISPR/Cas9 (AAV-CRISPR). The genetic deletion of Hmgcr by AAV-Cre resulted in extensive hepatocyte apoptosis and compensatory liver regeneration. At the biochemical level, we observed decreased levels of sterols and depletion of the nonsterol isoprenoids, dolichol and ubiquinone. At the cellular level, Hmgcr-null hepatocytes showed ER stress and impaired N-glycosylation. We further hypothesized that the depletion of dolichol, essential for N-glycosylation, could be responsible for ER stress. Using AAV-CRISPR, we somatically disrupted dehydrodolichyl diphosphate synthase subunit (Dhdds), encoding a branch point enzyme required for dolichol biosynthesis. Dhdds-null livers showed ER stress and impaired N-glycosylation, along with apoptosis and regeneration. Finally, the combined deletion of Hmgcr and Dhdds synergistically exacerbated hepatocyte ER stress. Our data show a critical role for mevalonate-derived dolichol in the liver and suggest that dolichol depletion is at least partially responsible for ER stress and apoptosis upon potent Hmgcr inhibition.