Effect of electrical impedance-guided PEEP in reducing pulmonary complications after craniotomy: study protocol for a randomized controlled trial.

OBJECTIVE: The purpose of this study is to explore whether electrical impedance tomography (EIT)-guided individualized positive end-expiratory pressure (PEEP) can reduce the incidence of pulmonary complications within 1 week following a craniotomy compared with a single PEEP (PEEP = 6 cmH2O) from dura suturing to extubation. METHODS: A randomized controlled trial will be conducted at the Second Affiliated Hospital of Soochou University. Five hundred forty patients undergoing a craniotomy in the supine position will be randomly allocated into the P6 (PEEP = 6 cmH2O) or Pi (individualized PEEP) group. Both groups of patients will receive a lung recruitment maneuver before suturing the dura. Then, the P6 group will receive 6 cmH2O PEEP, and the Pi group will receive EIT-guided individualized PEEP. The incidence and severity score of pulmonary complications within 1 week following surgery, the lung ultrasound score (LUS), regional cerebral oxygen saturation (rScO2), and PaO2/FiO2 before anesthesia (T0), 10 min after extubation (T1), 24 h after extubation (T2), and 72 h after extubation (T3) will be compared between the two groups. The duration of surgery and anesthesia, the level and duration of PEEP during surgery, the volume of liquid intake and output during surgery, and the postoperative ICU and hospital stays will be recorded. The main outcome of this study will be the incidence of pulmonary complications within 1 week after surgery. DISCUSSION: The purposes of this study are to determine whether EIT-guided individualized PEEP from the beginning of dura suturing to extubation reduces the incidence of pulmonary complications within 1 week after a craniotomy compared with a single constant PEEP and to evaluate the length of ICU and hospital stays. If our results are positive, this study will show that EIT-guided individualized PEEP is better than a single constant PEEP and can further improve the prognosis of neurosurgical patients and reduce hospitalization costs, which will promote the wide application of individualized PEEP in clinical anesthesia. TRIAL REGISTRATION: Chinese Clinical Trial Registry CHiCTR2100051200. Registered on 15 September 2021.

The complete mitochondrial genome of the Lethrinops lethrinus.

In the present study, for the first time, we reported the complete mitochondrial genome of Lethrinops lethrinus, which is 16,582 bp in length, including 13 protein-coding genes, 2 ribosomal RNA, 22 transfer RNA genes and a control zone. Moreover, its GC content is 45.99% (27.42% A, 26.59% T, 30.10% C, and 15.88% G), similar to that of Alticorpus geoffreyi (GC content of 45.82%). We further made the phylogenetic tree on the complete mitochondrial genomes of the above two species and other 12 closely related species to show their phylogenic relationship. The above results would facilitate our understanding of the evolution of Cichlidae mitochondrial genome.

The complete mitochondrial genome of the Placidochromis longimanus.

The Placidochromis longimanus (P. longimanus), one species of Cichlidae, resides in the Lake Malawi in East Africa. In present study, for the first time, we reported the complete mitochondrial genome of P. longimanus, which has 16 581 bp in length, including 13 protein-coding genes, 2 ribosomal RNA, 22 transfer RNA genes, and 1 control zone. Moreover, its GC content is 45.99% (27.44% A, 26.58% T, 30.11% C, and 15.87% G), similar to that of Astatotilapia calliptera (the GC content of 45.90%). We further made the phylogenetic tree on the complete mitochondrial genomes of the above two species and other 11 closely related species to show their phylogenic relationship. The above results would facilitate our understanding of the evolution of Cichlidae mitochondrial genome.

Neuroprotection of Cilostazol against ischemia/reperfusion-induced cognitive deficits through inhibiting JNK3/caspase-3 by enhancing Akt1.

Cilostazol(CTL) is a phosphodiesterase inhibitor, which has been widely used as anti-platelet agent. It also has preventive effects on various central nervous system (CNS) diseases, including ischemic stroke, Parkinson's disease and Alzheimer disease. However, the molecular mechanism underlying the protective effects of CTL is still unclear, and whether CTL can prevent I/R induced cognitive deficit has not been reported. Transient global brain ischemia was induced by 4-vessel occlusion in adult male Sprague-Dawley rats. The open field tasks and Morris water maze were used to assess the effect of CTL on anxiety-like behavioral and cognitive impairment after I/R. Western blotting were performed to examine the expression of related proteins, and HE-staining was used to detect the percentage of neuronal death in the hippocampal CA1 region. Here we found that CTL significantly improved cognitive deficits and the behavior of rats in Morris water maze and open field tasks (P<0.05). HE staining results showed that CTL could significantly protect CA1 neurons against cerebral I/R (P<0.05). Additionally, Akt1 phosphorylation levels were evidently up-regulated (P<0.05), while the activation of JNK3, which is an important contributor to I/R-induced neuron apoptosis, was reduced by CTL after I/R (P<0.05), and caspase-3 levels were also decreased by CTL treatment. Furthermore, all of CTL's protective effects were reversed by LY294002, which is a PI3K/Akt1 inhibitor. Taken together, our results suggest that CTL could protect hippocampal neurons and ameliorate the impairment of learning/memory abilities and locomotor/ exploratory activities in ischemic stroke via a PI3K-Akt1/JNK3/caspase-3 dependent mechanism.

The complete mitochondrial genome of the Fossorochromis rostratus.

The Fossorochromis rostratus is a species of cichlid endemic to Lake Malawi in East Africa. In this study, we first reported the complete mitochondrial genome of F. rostratus. The whole mitochondrial genome is 16 581 bp in length, which contains 13 protein-coding genes, 22 transfer RNA genes, and two ribosomal RNA genes. The GC content of this mitochondrial genome is 45.96% (27.47% A, 26.57%T, 30.12% C, and 15.84% G), similar to Astatotilapia calliptera (the GC content of 45.90%). We constructed a phylogenetic tree on the complete mitochondrial genomes of these two species and other 10 closely related species to show their phylogenic relationship. The complete mitochondrial genome of F. rostratus and its phylogenic relationship with other related species would facilitate our understanding of the evolution of Cichlidae mitochondrial genome.

The complete mitochondrial genome of the Alticorpus geoffreyi.

The present study reported the complete mitochondrial genome of Alticorpus geoffreyi for the first time. The mitochondrial genome of A. geoffreyi possesses 16,578 bp in length, involving 22 transfer RNA genes, 2 ribosomal RNA, 13 protein-coding genes and one control region. In addition, its GC content is 45.82% that is similar to that of Astatotilapia calliptera (the GC content of 45.90%). Based on the complete mitochondrial genomes of 14 closely related species, the phylogenetic tree was further made to show their phylogenic relationship. The results will serve as a useful dataset for studying the evolution of Cichlidae mitochondrial genome.

The complete mitochondrial genome of the Copadichromis mloto.

The present study reported the complete mitochondrial genome of Copadichromis mloto for the first time. The mitochondrial genome of C. mloto possesses 16,583bp in length, involving 22 transfer RNA genes, 2 ribosomal RNA, 13 protein-coding genes and a control region. In addition, its GC content is 45.94% that is similar to that of C. virginalis (the GC content of 45.74%). Based on the complete mitochondrial genomes of 13 closely related species, the phylogenetic tree was further made to show their phylogenic relationship. The results will serve as a useful dataset for studying the evolution of Cichlidae mitochondrial genome.

Hyperoxia-induced activation of the integrated stress response in the newborn rat lung.

Diverse environmental stresses stimulate eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, leading to a stress-resistant state characterized by global attenuation of protein synthesis and induction of cytoprotective genes. The signal transduction network culminating in these effects is referred to as the integrated stress response (ISR) or, when initiated by misfolded proteins within the endoplasmic reticulum (ER), the unfolded protein response (UPR). Given that we previously reported that exposure of 4-day-old Sprague-Dawley rats to 95% O(2) (Ox) diminishes global pulmonary protein synthesis and increases eIF2α phosphorylation, we conducted the current study to determine whether Ox activates the ISR or UPR. We found that Ox-induced alterations in ER morphology of alveolar type II cells and interstitial fibroblasts were not associated with activation of the UPR sensors PERK or activating transcription factor (ATF) 6 or with X-box binding protein-1 mRNA splicing in whole lung extracts. Exposure to Ox enhanced ATF4 immunoreactivity and nuclear protein content, followed by a 2- and 5-fold increase in ATF3 protein and mRNA expression, respectively. The accumulation of nuclear ATF4 protein coincided with induction of glutamate-cysteine ligase catalytic subunit, an ISR-responsive gene. Immunohistochemistry revealed that changes in ATF3/4 expression were prominent in the alveolus, whereas primary cell culture implicated epithelial and endothelial cells as targets. Finally, induction of ISR intermediates in the intact lung occurred in the absence of the phosphorylation of PKR, JNK, ERK1/2, and p38 MAPK. These findings demonstrate that Ox activates the ISR within the newborn lung and highlight regional and cell-specific alterations in the expression ISR transcription factors that regulate redox balance.

Hyperoxia inhibits protein synthesis and increases eIF2α phosphorylation in the newborn rat lung.

Prolonged exposure to hyperoxia contributes to aberrant lung growth in premature infants. Of the deleterious effects induced by hyperoxia, alterations in protein synthesis are likely to be of great importance to the developing lung. Regulation of mRNA translation occurs predominantly at the level of initiation via control of mRNA/ribosome binding by proteins known as eukaryotic initiation factors (eIF). Although hyperoxia is known to suppress mRNA translation in adult lungs, little is known regarding the effects in newborns or the involved mechanism. This study was performed to determine the effect of exposure to 95% O(2) on pulmonary protein synthesis in 4-day-old Sprague-Dawley rat pups. We found that hyperoxia suppressed the incorporation of [(3)H]phenylalanine into lung protein over time, resulting in a 23% reduction after 72 h compared with pups reared in room air. This effect was preceded by a shift in total lung RNA to lower order polysomes. Hyperoxia increased eIF4G-eIF4E binding, a surrogate maker of eIF4F complex assembly, and initially activated, then suppressed, the phosphorylation of ribosomal S6 kinase 1 and ribosomal S6 protein, downstream targets of mammalian target of rapamycin. Exposure to 95% O(2) enhanced the phosphorylation of the translational repressor eIF2α in whole lung extracts and the immunoreactivity of phosphorylated eIF2α in epithelial cells. Cell culture studies further demonstrated that hyperoxia increases eIF2α phosphorylation in lung epithelial cells, but not in lung fibroblasts. These findings illustrate that hyperoxia-induced suppression of mRNA translation in the newborn lung is accompanied by increased phosphorylation of eIF2α in the epithelium.

Hydrogen peroxide impairs insulin-stimulated assembly of mTORC1.

Oxidants are well recognized for their capacity to reduce the phosphorylation of the mammalian target of rapamycin (mTOR) substrates, eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and p70 S6 kinase 1 (S6K1), thereby hindering mRNA translation at the level of initiation. mTOR functions to regulate mRNA translation by forming the signaling complex mTORC1 (mTOR, raptor, GbetaL). Insulin signaling to mTORC1 is dependent upon phosphorylation of Akt/PKB and the inhibition of the tuberous sclerosis complex (TSC1/2), thereby enhancing the phosphorylation of 4E-BP1 and S6K1. In this study we report the effect of H(2)O(2) on insulin-stimulated mTORC1 activity and assembly using A549 and bovine aortic smooth muscle cells. We show that insulin stimulated the phosphorylation of TSC2 leading to a reduction in raptor-mTOR binding and in the quantity of proline-rich Akt substrate 40 (PRAS40) precipitating with mTOR. Insulin also increased 4E-BP1 coprecipitating with mTOR and the phosphorylation of the mTORC1 substrates 4E-BP1 and S6K1. H(2)O(2), on the other hand, opposed the effects of insulin by increasing raptor-mTOR binding and the ratio of PRAS40/raptor derived from the mTOR immunoprecipitates in both cell types. These effects occurred in conjunction with a reduction in 4E-BP1 phosphorylation and the 4E-BP1/raptor ratio. siRNA-mediated knockdown of PRAS40 in A549 cells partially reversed the effect of H(2)O(2) on 4E-BP1 phosphorylation but not on S6K1. These findings are consistent with PRAS40 functioning as a negative regulator of insulin-stimulated mTORC1 activity during oxidant stress.

Hyperoxia enhances VEGF release from A549 cells via post-transcriptional processes.

Exposure of animals to hyperoxia decreases lung VEGF mRNA expression concomitant with an acute increase in VEGF protein within the epithelial lining fluid (ELF). The VEGF concentration in ELF is in excess of that found in the plasma, leading to the hypothesis that hyperoxia stimulates the release of VEGF protein from stores within the extracellular matrix. To test this hypothesis in a cell culture system, we exposed A549 cells to 95% O(2) (Ox) for 48 h followed by recovery in room air (RA) for 24 h. We found that Ox increased VEGF protein two- to threefold within the medium at 48 h of exposure and during recovery. Heparin clearing revealed the medium to contain a 50/50 mixture of the heparin-binding (VEGF(165)) and heparin-nonbinding (VEGF(121)) proteins and that Ox increased both proteins equally. Transcriptional activation of VEGF seems unlikely to explain the increase in VEGF protein, as expression of full-length and splice variant VEGF mRNA was unchanged by hyperoxia. Analysis of cell-associated VEGF proteins found that Ox increased the expression of VEGF(121) and VEGF(165) proteins. Blocking binding sites with exogenous heparin enhanced VEGF protein in the medium from RA-grown cells, whereas heparinase digestion of bound VEGF revealed a greater reserve of VEGF protein in RA cells. Collectively these findings indicate that hyperoxia enhances the expression of VEGF(121/165) proteins and facilitates the release of VEGF(165) from cell-associated stores. Increases in VEGF in ELF may represent an adaptive response fostering cell survival and type II cell proliferation in O(2)-induced lung injury.

Roles of mitogen-activated protein kinase signal-integrating kinases 1 and 2 in oxidant-mediated eIF4E phosphorylation.

Oxidative stress alters cellular metabolic processes including protein synthesis. The eukaryotic initiation factor, eIF4E, acts in the rate-limiting steps of initiation and promotes nuclear export. Phosphorylation of eIF4E by mitogen activated protein kinase signal-integrating kinases 1 and 2 (Mnk) influences the affinity of eIF4E for the 5'-mRNA cap and fosters nuclear export activity. Although phosphorylation of eIF4E on Ser209 is observed following oxidant exposure, the contribution of Mnk isoforms and the significance of phosphorylation remain elusive. Using a Mnk inhibitor and fibroblasts derived from Mnk knockout mice, we demonstrate that that H2O2 enhances eIF4E phosphorylation in cells containing Mnk1. In contrast, cells containing only Mnk2 show little change or a decrease in eIF4E phosphorylation in response to H2O2. H2O2 also shifted eIF4GI protein from the nucleus to the cytoplasm suggesting that the increases in eIF4E phosphorylation may reflect enhanced substrate availability to cytoplasmic Mnk1. In Mnk1(+/+) cells, H2O2 also enhanced eIF4E phosphorylation in the nucleus to a greater degree than in the cytoplasm, an effect not observed in cells containing Mnk2. In response to H2O2, all MEFs showed increased eIF4E:4E-BP1 and 4E-BP2:eIF4E binding and reduced eIF4E:eIF4GI binding. We also observed a dramatic increase in the amount of Mnk1 associated with eIF4E following affinity chromatography. These changes coincided with a smaller reduction in global protein synthesis in response to H2O2 in the DKO cells. These findings suggest that changes in eIF4GI distribution may enhance eIF4E phosphorylation and that the presence of either Mnk1 or 2 or any degree of eIF4E phosphorylation negatively regulates global protein synthesis in response to oxidant stress.

Fiber specific differential phosphorylation of the alpha1-subunit of the Na(+),K (+)-ATPase in rat skeletal muscle: the effect of aging.

In skeletal muscle, the Na(+),K(+)-ATPase maintains the Na(+) and K(+) gradients and modulates contractile functions. The different fibers of the skeletal muscle possess diverse properties and functions, and thus, the demands for the Na-pump activity might be different. Because phosphorylation of the alpha1-subunit of the Na(+),K(+)-ATPase appears to serve a regulatory role in the activity of Na(+),K(+)-ATPase, we postulated that a difference in the phosphorylation of the alpha1-subunit may be found among the fibers. We utilized two well-characterized specific antibodies for the alpha1-subunit, namely the McK1 and alpha6F, to determine, by immunofluorescence, if the alpha1-subunit in rat skeletal muscle fiber is differentially phosphorylated. McK1 has the unique property that its binding to the alpha1-subunit is greatly reduced when Ser-18 is phosphorylated. Our data show that, in red gastrocnemius muscle, only a small number of the fibers were stained on the sarcolemmal membrane by McK1, while other fibers were almost completely devoid of any staining. By contrast, the staining pattern by McK1 in the white gastrocnemius muscle was mostly uniform. Immunostaining of serial sections using the alpha6F antibody showed that the alpha1-subunit is expressed in all fibers. Dephosphorylation of the tissue sections by phosphatase partially restored immunostaining of the alpha1-subunit by McK1. Fiber typing results showed that, in red gastrocnemius, those fibers stained positive for alpha1-subunit by McK1 are the Type I fibers, whereas those stained negative are the Type IIA, IID, and IIB fibers. With age, the number of fibers in red gastrocnemius stained positive for McK1 increased markedly in 30-month old rats compared to 6-month old rats. In conclusion, our result suggests that, in rats, the alpha1-subunit of the Na(+),K(+)-ATPase is differentially phosphorylated in the fibers of the red gastrocnemius muscle. Furthermore, advanced age is associated with an apparent decrease in the phosphorylation of the alpha1-subunit, in addition to the previously demonstrated increase in the levels of expression of the subunit.

Fiber type-specific immunostaining of the Na+,K+-ATPase subunit isoforms in skeletal muscle: age-associated differential changes.

The expression of the Na(+),K(+)-ATPase alpha and beta subunit isoforms in rat skeletal muscle and its age-associated changes have been shown to be muscle-type dependent. The cellular basis underlying these findings is not completely understood. In this study, we examined the expression of Na(+),K(+)-ATPase isoforms in individual fiber types and tested the hypothesis that, with age, the changes in the expression of the isoforms differ among individual fibers. We utilized immunohistochemical techniques to examine the expression of the subunit isoforms at the individual fiber levels. Immunofluorescence staining of the subunit isoforms in both white gastrocnemius (GW) and red gastrocnemius (GR) revealed a predominance of staining on the sarcolemmal membrane. Compared to the skeletal muscle of 6-month-old rats, there were substantial increases in the levels of alpha1, beta1, and beta3 subunit isoforms, and decreases in the levels of alpha2 and beta2 in 30-month-old rats. In addition, we found distinct patterns of staining for the alpha1, alpha2, beta1, and beta2 isoforms in tissue sections from young and aged rats. Muscle fiber-typing was performed to correlate the pattern of staining with specific fiber types. Staining for alpha1 and alpha2 isoforms in the skeletal muscle of young rats was generally evenly distributed among the fibers of GW and GR, with the exception of higher alpha1 levels in slow-twitch oxidative Type I fibers of GR. By contrast, staining for the beta1 and beta2 isoforms in the mostly oxidative fibers and the mostly glycolytic fibers, respectively, was almost mutually exclusive. With age, there was a fiber-type selective qualitative decrease of alpha2 and beta2 in Type IIB fibers, and increase of beta1 in Type IIB fibers and beta2 in Type IID fibers of white gastrocnemius. These results provide, at the individual fiber level, a cellular basis for the differential expression of the Na(+),K(+)-ATPase subunit isoforms in the muscle groups. The data further indicate that the aged-associated changes in expression of the subunit isoforms occur in both a fiber-type specific as well as an across fiber-type manner. Because of the differing biochemical properties of the subunit isoforms, these changes add another layer of complexity in our understanding of the adaptation of the Na-pump in skeletal muscle with advancing age.

Expression of phospholemman and its association with Na+-K+-ATPase in skeletal muscle: effects of aging and exercise training.

Phospholemman (PLM) is a recently identified accessory protein of the Na(+)-K(+)-ATPase (NKA), with a high level of expression in skeletal muscle. The objectives of this study are to characterize the PLM in skeletal muscle and to test the hypothesis that, as an accessory protein of NKA, expression of PLM and its association with the alpha-subunits of NKA is regulated during aging and with exercise training. PLM was characterized in skeletal muscle of 6- and 16-mo-old sedentary middle-aged rats (Ms), and the effects of aging and exercise training were studied in Ms, 29-mo-old sedentary senescent, and 29-mo-old treadmill-exercised senescent rats. Expression of PLM was muscle-type dependent, and immunofluorescence study showed that PLM distributed predominantly on the sarcolemmal membrane of the muscle fibers. Anti-PLM antibody reduced activity of NKA, and thus PLM appears to be required for NKA to express its full activity in skeletal muscle. Expression of PLM was not altered with aging but increased after exercise training. Coimmunoprecipitation studies demonstrated that PLM associates with both the alpha(1)- and alpha(2)-subunit isoforms of NKA. Compared with Ms rats, levels of PLM-associated alpha(1)-subunit increased in 29-mo-old sedentary senescent rats, and treadmill exercise has a tendency to partially reverse it. There was no significant change in PLM-associated alpha(2)-subunit with age, and exercise training has a tendency to increase that level. It is concluded that, in skeletal muscle, PLM appears to be a protein integral to the NKA complex and that PLM has the potential to modulate NKA in an isoform-specific and muscle type-dependent manner in aging and after exercise training.