Harumasa Nakazawa

Impact of fresh-frozen plasma from male-only donors versus mixed-sex donors on postoperative respiratory function in surgical patients: a prospective case-controlled study

BACKGROUND: To reduce the risk of transfusion‐related acute lung injury (TRALI), plasma products are mainly made from male donors in some countries because of the lower possibility of alloimmunization; other countries are considering this policy. The advantage of male‐only fresh‐frozen plasma (FFP) should be examined in a prospective case‐control study.

Inflammatory stimuli induce inhibitory S-nitrosylation of the deacetylase SIRT1 to increase acetylation and activation of p53 and p65

S-nitrosylation of the deacetylase SIRT1 functions as a proinflammatory switch in aging and inflammatory disorders. Flipping the SIRT1 Switch During Inflammation In aging-related diseases, chronic inflammation is associated with increased production of nitric oxide. Nitric oxide causes a posttranslational modification of proteins known as S-nitrosylation. SIRT1 is a protein deacetylase that inhibits the transcription factors p53 and NF-κB, which are involved in mediating cell death by apoptosis and promoting inflammatory responses. S-nitrosylation inhibits SIRT1 activity. Shinozaki et al. found that in cultured mammalian cells, S-nitrosylation of SIRT1 prevented it from deacetylating and inhibiting p53 and NF-κB. In mouse models of systemic inflammation, neurodegeneration, or muscle aging, S-nitrosylation of SIRT1 and the associated activation of p53 and NF-κB required the activity of nitric oxide synthases. Thus, S-nitrosylation of SIRT1 may be a critical factor in promoting apoptotic and inflammatory responses in aging-related diseases. Inflammation increases the abundance of inducible nitric oxide synthase (iNOS), leading to enhanced production of nitric oxide (NO), which can modify proteins by S-nitrosylation. Enhanced NO production increases the activities of the transcription factors p53 and nuclear factor κB (NF-κB) in several models of disease-associated inflammation. S-nitrosylation inhibits the activity of the protein deacetylase SIRT1. SIRT1 limits apoptosis and inflammation by deacetylating p53 and p65 (also known as RelA), a subunit of NF-κB. We showed in multiple cultured mammalian cell lines that NO donors or inflammatory stimuli induced S-nitrosylation of SIRT1 within CXXC motifs, which inhibited SIRT1 by disrupting its ability to bind zinc. Inhibition of SIRT1 reduced deacetylation and promoted activation of p53 and p65, leading to apoptosis and increased expression of proinflammatory genes. In rodent models of systemic inflammation, Parkinson’s disease, or aging-related muscular atrophy, S-nitrosylation of SIRT1 correlated with increased acetylation of p53 and p65 and activation of p53 and NF-κB target genes, suggesting that S-nitrosylation of SIRT1 may represent a proinflammatory switch common to many diseases and aging.

Effect of Cholecalciferol Supplementation on Vitamin D Status and Cathelicidin Levels in Sepsis: A Randomized, Placebo-controlled Trial

Objectives:To compare changes in vitamin D status and cathelicidin (LL-37) levels in septic ICU patients treated with placebo versus cholecalciferol. Design:Randomized, placebo-controlled, trial. Setting:Medical and surgical ICUs of a single teaching hospital in Boston, MA. Patients:Thirty adult ICU patients. Interventions:Placebo (n = 10) versus 200,000 IU cholecalciferol (n = 10) versus 400,000 IU cholecalciferol (n = 10), within 24 hours of new-onset severe sepsis or septic shock. Measurements and Main Results:Blood samples were obtained at baseline (day 1) and on days 3, 5, and 7, to assess total 25-hydroxyvitamin D, as well as vitamin D–binding protein and albumin to calculate bioavailable 25-hydroxyvitamin D. Plasma LL-37 and high-sensitivity C-reactive protein levels were also measured. At baseline, median (interquartile range) plasma 25-hydroxyvitamin D was 17 ng/mL (13–22 ng/mL) and peaked by day 5 in both intervention groups. Groups were compared using Kruskal-Wallis tests. Relative to baseline, on day 5, median change in biomarkers for placebo, 200,000 IU cholecalciferol, and 400,000 IU cholecalciferol groups, respectively, were as follows: 1) total 25-hydroxyvitamin D, 3% (–3% to 8%), 49% (30–82%), and 69% (55–106%) (p < 0.001); 2) bioavailable 25-hydroxyvitamin D, 4% (–8% to 7%), 45% (40–70%), and 96% (58–136%) (p < 0.01); and 3) LL-37: –17% (–9% to –23%), 4% (–10% to 14%), and 30% (23–48%) (p = 0.04). Change in high-sensitivity C-reactive protein levels did not differ between groups. A positive correlation was observed between bioavailable 25-hydroxyvitamin D and LL-37 (Spearman &rgr; = 0.44; p = 0.03) but not for total 25-hydroxyvitamin D and LL-37. Conclusions:High-dose cholecalciferol supplementation rapidly and safely improves 25-hydroxyvitamin D and bioavailable 25-hydroxyvitamin D levels in patients with severe sepsis or septic shock. Changes in bioavailable 25-hydroxyvitamin D are associated with concomitant increases in circulating LL-37 levels. Larger trials are needed to verify these findings and to assess whether optimizing vitamin D status improves sepsis-related clinical outcomes.

Battery of behavioral tests in mice to study postoperative delirium.

Postoperative delirium is associated with increased morbidity, mortality and cost. However, its neuropathogenesis remains largely unknown, partially owing to lack of animal model(s). We therefore set out to employ a battery of behavior tests, including natural and learned behavior, in mice to determine the effects of laparotomy under isoflurane anesthesia (Anesthesia/Surgery) on these behaviors. The mice were tested at 24 hours before and at 6, 9 and 24 hours after the Anesthesia/Surgery. Composite Z scores were calculated. Cyclosporine A, an inhibitor of mitochondria permeability transient pore, was used to determine potential mitochondria-associated mechanisms of these behavioral changes. Anesthesia/Surgery selectively impaired behaviors, including latency to eat food in buried food test, freezing time and time spent in the center in open field test, and entries and duration in the novel arm of Y maze test, with acute onset and various timecourse. The composite Z scores quantitatively demonstrated the Anesthesia/Surgery-induced behavior impairment in mice. Cyclosporine A selectively ameliorated the Anesthesia/Surgery-induced reduction in ATP levels, the increases in latency to eat food, and the decreases in entries in the novel arm. These findings suggest that we could use a battery of behavior tests to establish a mouse model to study postoperative delirium.

iNOS as a Driver of Inflammation and Apoptosis in Mouse Skeletal Muscle after Burn Injury: Possible Involvement of Sirt1 S-Nitrosylation-Mediated Acetylation of p65 NF-κB and p53

Inflammation and apoptosis develop in skeletal muscle after major trauma, including burn injury, and play a pivotal role in insulin resistance and muscle wasting. We and others have shown that inducible nitric oxide synthase (iNOS), a major mediator of inflammation, plays an important role in stress (e.g., burn)-induced insulin resistance. However, it remains to be determined how iNOS induces insulin resistance. Moreover, the interrelation between inflammatory response and apoptosis is poorly understood, although they often develop simultaneously. Nuclear factor (NF)-κB and p53 are key regulators of inflammation and apoptosis, respectively. Sirt1 inhibits p65 NF-κB and p53 by deacetylating these transcription factors. Recently, we have shown that iNOS induces S-nitrosylation of Sirt1, which inactivates Sirt1 and thereby increases acetylation and activity of p65 NF-κB and p53 in various cell types, including skeletal muscle cells. Here, we show that iNOS enhances burn-induced inflammatory response and apoptotic change in mouse skeletal muscle along with S-nitrosylation of Sirt1. Burn injury induced robust expression of iNOS in skeletal muscle and gene disruption of iNOS significantly inhibited burn-induced increases in inflammatory gene expression and apoptotic change. In parallel, burn increased Sirt1 S-nitrosylation and acetylation and DNA-binding capacity of p65 NF-κB and p53, all of which were reversed or ameliorated by iNOS deficiency. These results indicate that iNOS functions not only as a downstream effector but also as an upstream enhancer of burn-induced inflammatory response, at least in part, by Sirt1 S-nitrosylation-dependent activation (acetylation) of p65 NF-κB. Our data suggest that Sirt1 S-nitrosylation may play a role in iNOS-mediated enhanced inflammatory response and apoptotic change, which, in turn, contribute to muscle wasting and supposedly to insulin resistance after burn injury.

Impact of immunoreactive substances contained in apheresis platelet concentrate on postoperative respiratory function in surgical patients receiving platelet transfusion: a prospective cohort study.

To construct an alternative policy for the donor selection of platelet concentrate (PC), a clinical study exploring the features of lung injury following PC administration is needed.

Low‐Dose Farnesyltransferase Inhibitor Suppresses HIF‐1α and Snail Expression in Triple‐Negative Breast Cancer MDA‐MB‐231 Cells In Vitro

The aggressiveness of triple‐negative breast cancer (TNBC), which lacks estrogen receptor, progesterone receptor and epidermal growth factor receptor 2 (HER2), represents a major challenge in breast cancer. Migratory and self‐renewal capabilities are integral components of invasion, metastasis and recurrence of TNBC. Elevated hypoxia‐inducible factor‐1α (HIF‐1α) expression is associated with aggressiveness of cancer. Nonetheless, how HIF‐1α expression is regulated and how HIF‐1α induces aggressive phenotype are not completely understood in TNBC. The cytotoxic effects of farnesyltransferase (FTase) inhibitors (FTIs) have been studied in cancer and leukemia cells. In contrast, the effect of FTIs on HIF‐1α expression has not yet been studied. Here, we show that clinically relevant low‐dose FTI, tipifarnib (300 nM), decreased HIF‐1α expression, migration and tumorsphere formation in human MDA‐MB‐231 TNBC cells under a normoxic condition. In contrast, the low‐dose FTIs did not inhibit cell growth and activity of the Ras pathway in MDA‐MB 231 cells. Tipifarnib‐induced decrease in HIF‐1α expression was associated with amelioration of the Warburg effect, hypermetabolic state, increases in Snail expression and ATP release, and suppressed E‐cadherin expression, major contributors to invasion, metastasis and recurrence of TBNC. These data suggest that FTIs may be capable of ameliorating the aggressive phenotype of TNBC by suppressing the HIF‐1α‐Snail pathway. J. Cell. Physiol. 232: 192–201, 2017.

Role of Protein Farnesylation in Burn-Induced Metabolic Derangements and Insulin Resistance in Mouse Skeletal Muscle

Objective Metabolic derangements, including insulin resistance and hyperlactatemia, are a major complication of major trauma (e.g., burn injury) and affect the prognosis of burn patients. Protein farnesylation, a posttranslational lipid modification of cysteine residues, has been emerging as a potential component of inflammatory response in sepsis. However, farnesylation has not yet been studied in major trauma. To study a role of farnesylation in burn-induced metabolic aberration, we examined the effects of farnesyltransferase (FTase) inhibitor, FTI-277, on burn-induced insulin resistance and metabolic alterations in mouse skeletal muscle. Methods A full thickness burn (30% total body surface area) was produced under anesthesia in male C57BL/6 mice at 8 weeks of age. After the mice were treated with FTI-277 (5 mg/kg/day, IP) or vehicle for 3 days, muscle insulin signaling, metabolic alterations and inflammatory gene expression were evaluated. Results Burn increased FTase expression and farnesylated proteins in mouse muscle compared with sham-burn at 3 days after burn. Simultaneously, insulin-stimulated phosphorylation of insulin receptor (IR), insulin receptor substrate (IRS)-1, Akt and GSK-3β was decreased. Protein expression of PTP-1B (a negative regulator of IR-IRS-1 signaling), PTEN (a negative regulator of Akt-mediated signaling), protein degradation and lactate release by muscle, and plasma lactate levels were increased by burn. Burn-induced impaired insulin signaling and metabolic dysfunction were associated with increased inflammatory gene expression. These burn-induced alterations were reversed or ameliorated by FTI-277. Conclusions Our data demonstrate that burn increased FTase expression and protein farnesylation along with insulin resistance, metabolic alterations and inflammatory response in mouse skeletal muscle, all of which were prevented by FTI-277 treatment. These results indicate that increased protein farnesylation plays a pivotal role in burn-induced metabolic dysfunction and inflammatory response. Our study identifies FTase as a novel potential molecular target to reverse or ameliorate metabolic derangements in burn patients.

FRET-based evaluation of Bid cleavage in a single primary cultured neuron.

Apoptosis is a cell death modality that is initiated by the activation of caspases. Theoretically, fluorescence resonance energy transfer (FRET) analysis should be a convenient tool for visualizing the activation of caspase. Since the FRET probe cannot be transfected in primary neuronal cultures effectively, the FRET signal is not sufficiently strong for evaluations. We developed a method of extracting the significant signals from the fluorescent FRET images that enables the initiation of apoptosis to be analyzed. We used primary hippocampal cultures transfected with a vector encoding Bid fused with YFP and CFP. Apoptosis was induced using staurosporine (STS; 1μM). The CFP and YFP signals were observed using an inverted fluorescence microscope and were processed using imaging software for analysis. After the background signal was subtracted, the area of caspase activation and the significant signals were extracted from the localized intense signals originating from mitochondria. The CFP and YFP intensities of a selected area in a single neuron were integrated, and the CFP/YFP ratio was obtained. To confirm caspase activation in a similar experimental setting, a luminescence analysis was also performed. The FRET signals from the cultured neuron were confined to foci, since the Bid linker was specifically localized in the mitochondria. The extracted CFP and YFP signals from the foci were strong enough to be evaluated. The average CFP/YFP ratio in the neuron increased significantly after an STS challenge, from 0.673±0.024 (control) to 1.008±0.134 (STS) (mean±SD) (P<0.05). Our study demonstrated, for the first time, the quantification of Bid cleavage as expressed by FRET in a primary neuron. Since Bid is localized in the mitochondria, the region of interest was restricted to a specific area, enabling the signal to be analyzed. This methodology may be useful for the application of FRET analyses in primary cultured cells.

Burn-induced muscle metabolic derangements and mitochondrial dysfunction are associated with activation of HIF-1α and mTORC1: Role of protein farnesylation

Metabolic derangements are a clinically significant complication of major trauma (e.g., burn injury) and include various aspects of metabolism, such as insulin resistance, muscle wasting, mitochondrial dysfunction and hyperlactatemia. Nonetheless, the molecular pathogenesis and the relation between these diverse metabolic alterations are poorly understood. We have previously shown that burn increases farnesyltransferase (FTase) expression and protein farnesylation and that FTase inhibitor (FTI) prevents burn-induced hyperlactatemia, insulin resistance, and increased proteolysis in mouse skeletal muscle. In this study, we found that burn injury activated mTORC1 and hypoxia-inducible factor (HIF)-1α, which paralleled dysfunction, morphological alterations (i.e., enlargement, partial loss of cristae structure) and impairment of respiratory supercomplex assembly of the mitochondria, and ER stress. FTI reversed or ameliorated all of these alterations in burned mice. These findings indicate that these burn-induced changes, which encompass various aspects of metabolism, may be linked to one another and require protein farnesylation. Our results provide evidence of involvement of the mTORC1-HIF-1α pathway in burn-induced metabolic derangements. Our study identifies protein farnesylation as a potential hub of the signaling network affecting multiple aspects of metabolic alterations after burn injury and as a novel potential molecular target to improve the clinical outcome of severely burned patients.