CLK2 mediates IκBα-independent early termination of NF-κB activation by inducing cytoplasmic redistribution and degradation.

Activation of the NF-κB pathway is strictly regulated to prevent excessive inflammatory and immune responses. In a well-known negative feedback model, IκBα-dependent NF-κB termination is a delayed response pattern in the later stage of activation, and the mechanisms mediating the rapid termination of active NF-κB remain unclear. Here, we showed IκBα-independent rapid termination of nuclear NF-κB mediated by CLK2, which negatively regulated active NF-κB by phosphorylating the RelA/p65 subunit of NF-κB at Ser180 in the nucleus to limit its transcriptional activation through degradation and nuclear export. Depletion of CLK2 increased the production of inflammatory cytokines, reduced viral replication and increased the survival of the mice. Mechanistically, CLK2 phosphorylated RelA/p65 at Ser180 in the nucleus, leading to ubiquitin‒proteasome-mediated degradation and cytoplasmic redistribution. Importantly, a CLK2 inhibitor promoted cytokine production, reduced viral replication, and accelerated murine psoriasis. This study revealed an IκBα-independent mechanism of early-stage termination of NF-κB in which phosphorylated Ser180 RelA/p65 turned off posttranslational modifications associated with transcriptional activation, ultimately resulting in the degradation and nuclear export of RelA/p65 to inhibit excessive inflammatory activation. Our findings showed that the phosphorylation of RelA/p65 at Ser180 in the nucleus inhibits early-stage NF-κB activation, thereby mediating the negative regulation of NF-κB.

Insights into water freezing from classical nucleation theory.

Water freezing is a crucial physical phenomenon. The process of ice formation, and the estimation of the ice nucleation rate also have important applications. However, until now, the experimental phenomenon of rapid freezing of water in nanoseconds has not been fully explained theoretically, and the physics underlying the experimental phenomenon has still not been revealed. In this work, combining classical nucleation theory with Mie theory, a kinetic model is developed that reproduces for the first time the experimental phenomenon of decreasing transmissivity. The process of ice formation (nucleation, growth and engulfment) has been revealed. In the process of theoretical derivation, the Zel'dovich-Frenkel (ZF) equation is developed, indicating a limit to the phase transition driving force |Δµ|/(kBT) ≤ 1. By analyzing the experimental and simulation results, it is suggested that the change in the transparency of the sample may be caused by the ice/vacuum interface scattering. In addition, during the rapid phase transition, it was found that the phase transition continues to occur even after phase fraction normalization. Finally, the approximate formula between the nucleation rate and sample transparency is given. This formula can predict the change of sample transparency during phase transition and provides a way to measure the nucleation rate. The results presented here give an insight into the phase transition kinetics, and the methodology may also work for the phase transitions of other materials.

Phosphorylation of MAVS/VISA by Nemo-like kinase (NLK) for degradation regulates the antiviral innate immune response.

MAVS is essential for antiviral immunity, but the molecular mechanisms responsible for its tight regulation remain poorly understood. Here, we show that NLK inhibits the antiviral immune response during viral infection by targeting MAVS for degradation. NLK depletion promotes virus-induced antiviral cytokine production and decreases viral replication, which is potently rescued by the reintroduction of NLK. Moreover, the depletion of NLK promotes antiviral effects and increases the survival times of mice after infection with VSV. NLK interacts with and phosphorylates MAVS at multiple sites on mitochondria or peroxisomes, thereby inducing the degradation of MAVS and subsequent inactivation of IRF3. Most importantly, a peptide derived from MAVS promotes viral-induced IFN-ß production and antagonizes viral replication in vitro and in vivo. These findings provide direct insights into the molecular mechanisms by which phosphorylation of MAVS regulates its degradation and influences its activation and identify an important peptide target for propagating antiviral responses.

Assessment of Pain Associated with the Injection of Sodium Pentobarbital in Laboratory Mice (Mus musculus).

The AVMA Guidelines for the Euthanasia of Animals considers injection of barbiturates to be an acceptable method of euthanasia in rodents but states there is a potential for pain when administered intraperitoneally. This study examined the potential for pain in mice by assessing visceral pain after intraperitoneal administration and acute pain by using a paw-lick test. Male and female mice (n = 160) intraperitoneally received a euthanizing dose of sodium pentobarbital at a concentration of 5, 50, or 390 mg/mL and were observed for writhing, peritoneum-directed behaviors (PDB), loss of righting reflex, and time to death. Writhing was not observed in any animal. There was no significant difference in the number of mice exhibiting PDB or in the rate of PDB for responders receiving either saline or the 390-mg/mL solution. There was a significant treatment effect on time, with greater concentration and dose resulting in more rapid loss of righting reflex and death. In the second set of experiments, the same solutions were injected subcutaneously into the plantar hindpaw of male and female mice (n = 84). The number of responders, latency until the first lick, and the number of licks per responder were recorded. The number of responders was increased in the 50-mg/mL group; however, there was no difference in latency or the number of licks per responder. These results show that intraperitoneal injection of sodium pentobarbital for euthanasia in mice did not result in increased behavioral signs of pain, and animals lose consciousness more rapidly than the onset of pain seen in the pawlick test. Therefore, although sodium pentobarbital is capable of inducing inflammation, euthanasia through intraperitoneal administration is rapid and does not result in overt signs of pain when compared with injection of saline.

[Experimental study on prevention of diabetes complicated with tuberculosis by Mycobacterium vaccae].

OBJECTIVE: To study if Mycobacterium vaccae has any effect in prevention of rats with diabetes from tuberculosis. METHODS: Wistar rats with diabetes which were produced by injection of streptozotocin one week before were divided into four groups. Mycobacterium vaccae was injected into Wistar rats with diabetes and one month later H(37)R(v) was injected intravenously. The rats were sacrificed six weeks later to observe pathomorphologic changes in lung tissues. Indexes for evaluation included quantitative culture of Mycobacterium tuberculosis of lung tissue, tubercular lesion, count of Mycobacterium tuberculosis in alveolar macrophages and histopathological changes. RESULTS: Pathological change index of tubercular lesion, quantitative culture of Mycobacterium tuberculosis in lung tissue, count of Mycobacterium tuberculosis in alveolar macrophages were 2.5 +/- 0.7, (4.1 +/- 0.6) x 10(4) cfu, 2.6 +/- 0.9 respectively in group injected with Mycobacterium vaccae, while 3.3 +/- 0.5, (9.9 +/- 1.0) x 10(4) cfu, 7.2 +/- 0.7 respectively in the control group. Statistically significant differences were found between the two groups (P < 0.05, < 0.01, < 0.01 respectively). The histopathological changes of lung tissue in group of diabetes complicated with tuberculosis revealed that the lesions were predominant with necrosis, while in group injected with Mycobacterium vaccae, the lesions showed proliferation mainly. This indicated that Mycobacterium vaccae could decrease the damage to the lung tissue of the rat with diabetes by tubercle bacilli. CONCLUSION: Mycobacterium vaccae might play a role in prevention of rat with diabetes from tuberculosis.