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The primers that were used to amplify the 3′UTR of candidate genes are available upon request. 3′UTRs of genes of interest were cloned into the modified pPD129.57 vector as described previously (18). The data for 3′UTR expression and for VPC timing were analyzed using χ2 test.
To determine viability, 20-μL aliquots (60–100 worms) were placed every 3 d onto two 6-cm nematode growth medium (NGM) plates seeded with OP50, and the numbers of L1 worms were recorded as number of plated worms (Np). A total of 16–24 h later, the density of newly hatched L1 worms was adjusted to three to five worms per microliter S-basal. The eggs were transferred to plates seeded with HB101 and bleached again 3 d later. Briefly, worms were well fed for at least two generations, and gravid adults were bleached with hypochlorite and sodium hydroxide. L1 starvation assay was adapted from a previously described protocol (3). Worms strains were grown and maintained at 20 °C as described (29).

Enabling Duo Restore (Updated)

For example, those objective circumstances could be linked to inflation, shortages in the supply chain or the fact that there is a better alternative to fulfil the intended policy objective of a measure. The backups are encrypted by the recovery password, which is only known to you and cannot be recovered by Duo. If you manually delete accounts within the app then they are gone and there is no process for restoration.
SMART Takes is a monthly newsletter filled with content about self-empowered, practical, and evidence-informed recovery. All proceeds go to support our fight against the addiction epidemic in America. DALLAS (FWAA) – Memphis is the winner of the Cheez-It Crunch Time Play of the Week for Week 4 after safety Chris Bracy’s fourth-quarter strip and recovery sealed an 18-point comeback win over Arkansas, as selected by the All-America Commitee of the Football Writers Association of America. NACTO connects and mobilizes North American cities and transit agencies toward safe, sustainable, and accessible transportation. Elegans Genetic Center for many mutants of miRNA and other genes. For examining the age-1 3′UTR reporter, revery play login the rol-6(d) marker (100 ng/μl pRF4) was used instead of the unc-119(+) plasmid.

Associated Data

When late, first larval stage (L1) worms sense unfavorable conditions, they enter an alternative and long-lived larval stage called dauer larvae (or dauer diapause). The nematode Caenorhabditis elegans responds to starvation by entering developmental arrest at multiple stages of its life cycle (1). Extreme climate events such as droughts and heatwaves are intensifying under climate change, yet their combined effects on plant recovery remain unclear. These pages contain all relevant country-specific information, including the recovery and resilience plans, the Commission’s assessment of the plans as well as information on payments requested by the Member States and funds paid out by the Commission.
Furthermore, worms that are long-lived due to dietary restriction or decreased mitochondrial respiratory rates are short-lived during L1 diapause, suggesting that the mechanisms controlling L1 starvation survival are different at least in some aspects from those controlling aging (3). We provide evidence that miRNA miR-71 is not required for the animals’ entry into L1 diapause, but plays a critical role in long-term survival by repressing the expression of insulin receptor/PI3K pathway genes and genes acting downstream or in parallel to the pathway. Full device encrypted backups to iTunes will back up both the account listings and private key pairs, but can only be restored on the SAME phone that created the backup. If you enabled third-party account backup, you can recover your accounts on your iOS or Android device. If your organization hasn’t enabled self-service device management, contact your IT Help Desk or Duo service administrator for assistance reactivating the account.
The RRF funding amounts shown for measures are based on the initial cost estimates provided by the Member States included in the recovery and resilience plans and may as such differ from finalized Commission financial reports, which follow eventual project implementation. The funding amounts shown reflect the initial cost estimates included in the national recovery and resilience plans. Member States use the funds provided by the Recovery and Resilience Facility to implement ambitious reforms and investment to make their economies and societies more sustainable, resilient and prepared for the green and digital transitions. The Scoreboard gives an overview of progress in implementing the Facility and the national recovery and resilience plans.

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On the other hand, the role of a particular miRNA (miR-71) is executed by repressing the expression of many genes in multiple pathways. On one hand, we showed that deletions of a good number of miRNAs have varying impacts on the L1 diapause survival rate, although they may effect the rate through different mechanisms. Instead, many specific physiological functions, such as the starvation-induced stress response, are regulated by a miRNA-target network, often involving multiple miRNAs and a large number of their targets. We found that the known developmental timing genes, hbl-1, lin-42, and lit-1, were at the top of the list (TargetScan). To understand how miR-71 affects VPC division, we searched its predicted targets for potential genes involved in regulating developmental timing. These results indicate that miR-71 plays a significant role in larval development of animals recovering from L1 diapause and likely does so by regulating the expression of components of the insulin receptor/DAF-16 pathway, as well as factors acting downstream, or in parallel to, DAF-16.

Recovering Duo-Protected Accounts with Instant Restore (Updated)

If you created a Google Drive backup using the old Duo Restore toggle and know the recovery password you set when enabling Duo Mobile backup, you can still access this backup to perform an Instant Restore. If you have Google account backup disabled or your device does not have a PIN, pattern, or password screen lock, please see Enabling Duo Restore for the legacy Instant Restore flow. You’ll still need to perform the Instant Restore steps before you can use those accounts to log in to Duo-protected services with Duo Push or Duo Mobile passcodes. Note that this doesn’t reconnect your Duo-protected accounts.

(H) Fluorescence and DIC images showing that a lin-42 3′UTR reporter was repressed in mir-71(+) worms (2/2 transgenic lines) and prominently derepressed in mir-71(−) worms (2/2 transgenic lines).
(H and I) Fluorescence images (H) and statistical data (I) showing that the M cell diveded in fed animals but remained undivided in 4-, 7-, or 11-d–starved L1 wild-type and mir-71(lf) worms.
The Scoreboard gives an overview of progress in implementing the Facility and the national recovery and resilience plans.
The RRF is also crucial for implementing the REPowerEU plan – the Commission’s response to the socio-economic hardships and global energy market disruption caused by Russia’s invasion of Ukraine.
These pages contain all relevant country-specific information, including the recovery and resilience plans, the Commission’s assessment of the plans as well as information on payments requested by the Member States and funds paid out by the Commission.
If you have Google account backup disabled or your device does not have a PIN, pattern, or password screen lock, please see Enabling Duo Restore for the legacy Instant Restore flow.

Whereas the vulva of wild-type worms developed into the pyramidal stage (81 of 82 worms), the P6.p of mir-71(n4115, lf) mutant worms divided only once (83 of 89 worms). The computation-based prediction that age-1 and pdk-1 are potential targets of miR-71 was also reported in a recent study focusing on miRNA functions in aging where the mRNA level of pdk-1 was shown to be up-regulated in mir-71 worms (14). (C) Fluorescence and differential interference contrast (DIC) images showing that the age-1 3′UTR reporter was repressed in mir-71(+) worms (3/4 transgenic lines) but not in mir-71(lf) worms (4/4 transgenic lines). The transcript level of unc-31 was increased in mir-71(lf) worms, compared with that of wild-type controls that were normalized to the value of 1. MiR-71 represses the expression of age-1 and unc-31 through the actions on their 3′UTR, but miR-71 is not required for arresting M cell division during L1 diapause.

To use Instant Restore, you must have previously included Duo Mobile app data in your Android device backup.
The funding amounts shown reflect the initial cost estimates included in the national recovery and resilience plans.
If you created a Google Drive backup using the old Duo Restore toggle and know the recovery password you set when enabling Duo Mobile backup, you can still access this backup to perform an Instant Restore.
Individual GFP reporter constructs for candidate genes (4 ng/μL) and the mCherry internal control plasmid (4 ng/μL) were mixed with unc-119 rescuing plasmid (20 ng/μL) and pBluescript KS+ (72 ng/μL) and coinjected into unc-119(ed3) and mir-71(n4115); unc-119(ed3) worms following standard protocols (32).
Elegans and Caenorhabditis briggsae, leading us to focus further analyses on these two genes.
As pointed out above, multiple miRNAs in addition to miR-71 and the let-7 family miRNAs have roles in L1 diapause, and they may regulate the expression of many diverse targets that may include, but are not limited to, factors involved in UNC-31–InsR-signaling activities.

Here we show that compromising overall microRNA (miRNA) functions or mutating certain individual miRNAs impairs the long-term survival of nematodes during starvation-induced L1 diapause. Third-party accounts will also be restored if third-party backup was enabled on the old device. If you become locked out of those services and don’t have a backup of your accounts in Duo Mobile, you’ll need to contact the support team for that application (or perform the account recovery process for each of those third-party applications). However, whether an account can be restored depends upon Duo Restore being enabled by the administrator in the Duo Admin Panel or whether you’ve set a recovery password for reconnecting third-party accounts.
That’s why I’m determined to continue delivering more council homes as we build a fairer London for everyone. A real pleasure to meet George Clarke and hear about his experiences growing up on a council estate. That’s why our recovery experts provide a custom treatment plan to fit each individual’s circumstances. You’ve taken the first step on your path to recovery. Images were pseudocolored in Photoshop CS3 (Adobe) and assembled in Illustrator CS3 (Adobe).
{We further examined worms recovering from 4 d of L1 starvation and found that around 90% of the mir-71(lf) mutants displayed retarded vulval precursor cell (VPC) division, compared with less than 5% in wild type (Fig. 4A). We found that the 3′UTRs of several genes of the InsR pathway, including unc-31, age-1, pdk-1, akt-2, and sgk-1, contain predicted miR-71 targeting sites (as predicted by TargetScan and mirWIP). (H and I) Fluorescence images (H) and statistical data (I) showing that the M cell diveded in fed animals but remained undivided in 4-, 7-, or 11-d–starved L1 wild-type and mir-71(lf) worms. (E) Fluorescence and DIC images showing that the unc-31 3′UTR reporter was repressed in mir-71(+)worms (2/2 transgenic lines) but not in mir-71(lf) worms (4/4 transgenic lines). We found that the poor survival rate of daf-16(mu86)(lf) was further decreased by mir-71(lf) (Fig. 2C), consistent with the notion that a portion of miR-71 activities regulate genes that act in parallel to UNC-31–mediated InsR/PI3K signaling for long-term survival during L1 diapause. Mutating miR-71 drastically reduces the survival rate of animals in L1 diapause, and the effect can be suppressed by mutations of insulin receptor pathway genes age-1 and unc-31.}