We conducted a comprehensive time-resolved resonance Raman spectroscopy study of the structures of the retinal chromophore during the photocycle of the sodium-ion pump Krokinobacter rhodopsin 2 (KR2). We succeeded in determining the structure of the chromophore in the unphotolyzed state and in the K, L, M, and O intermediates, by overcoming the problem that only a small fraction of the M intermediate is accumulated in the KR2 photocycle. The Schiff base in the retinal chromophore forms a strong hydrogen bond in the unphotolyzed state and in the K, L, and O intermediates and is deprotonated in the M intermediate. Formation of this strong hydrogen bond facilitates deprotonation of the Schiff base, which is necessary for the sodium ion to move past the Schiff base. The polyene chain in the chromophore of KR2 is twisted in all of the states of the photocycle: the portion near the Schiff base is largely twisted in the unphotolyzed state and in the K intermediate, whereas the middle portion of the polyene chain becomes largely twisted in the L, M, and O intermediates. During the photocycle, the twisted structure of the polyene chain and strong hydrogen bond at the Schiff base are advantageous for transient relocation of the Schiff base proton. The obtained resonance Raman data clarified the unique structural features of the KR2 chromophore, which are not accessible by other methods.

BackgroundLactate levels within tumors are correlated with metastases, tumor recurrence, and radioresistance, thus apparently contributing to poor outcomes in patients with various cancers. We previously reported that high-level production of lactate by multiple myeloma (MM) cell lines is associated with high-level LDH activity within such MM cells. However, the kinetics of lactate remains to be studied. In the present study, we attempted to elucidate the mechanism of lactate incorporation into MM cells.MethodsSix MM cell lines and stromal cells obtained through long-term culture of bone marrow samples from MM patients were employed. Incorporation of lactate was quantified using C14-labeled lactate. The role of MCT1, a member of the monocarboxylate transporters (MCTs), expressed on MM cells, was examined in the presence of its inhibitor (α-cyano-4-hydroxycinnamic acid: CHC) and by using gene-silencing technique.ResultsMM cell lines as well as stromal cells were found to produce lactate. Incorporation of C14-labeled lactate into MM cells occurred in all 6 MM cell lines analyzed. Inhibition of MCT1 by using CHC or MCT1-targeting siRNA reduced lactate incorporation and caused apoptosis in MM cells. This apoptosis was enhanced when the activity of pyruvate dehydrogenase kinase was blocked by dichroloacetate. Survival of normal peripheral blood mononuclear cells was not influenced by MCT1 inhibition.ConclusionsThe present data suggest that lactate is produced by MM cell lines and stromal cells, and contributes to the survival of such MM cells in autocrine or paracrine manners. Suppression of lactate incorporation by targeting MCT1 may provide a novel therapeutic strategy for MM which may be applicable for other B-cell neoplasms.

p97/VCP is an endoplasmic reticulum (ER)‐associated protein that belongs to the AAA (ATPases associated with diverse cellular activities) ATPase family. It has a variety of cellular functions including ER‐associated protein degradation, autophagy, and aggresome formation. Recent studies have shown emerging roles of p97/VCP and its potential as a therapeutic target in several cancer subtypes including multiple myeloma (MM). We conducted a cell‐based compound screen to exploit novel small compounds that have cytotoxic activity in myeloma cells. Among approximately 2000 compounds, OSSL_325096 showed relatively strong antiproliferative activity in MM cell lines (IC50, 100‐500 nmol/L). OSSL_325096 induced apoptosis in myeloma cell lines, including a bortezomib‐resistant cell line and primary myeloma cells purified from patients. Accumulation of poly‐ubiquitinated proteins, PERK, CHOP, and IREα, was observed in MM cell lines treated with OSSL_325096, suggesting that it induces ER stress in MM cells. OSSL_325096 has a similar chemical structure to DBeQ, a known p97/VCP inhibitor. Knockdown of the gene encoding p97/VCP induced apoptosis in myeloma cells, accompanied by accumulation of poly‐ubiquitinated protein. IC50 of OSSL_325096 to myeloma cell lines were found to be lower (0.1‐0.8 μmol/L) than those of DBeQ (2‐5 μmol/L). In silico protein–drug‐binding simulation suggested possible binding of OSSL_325096 to the ATP binding site in the D2 domain of p97/VCP. In cell‐free ATPase assays, OSSL_325096 showed dose‐dependent inhibition of p97/VCP ATPase activity. Finally, OSSL_325096 inhibited the growth of subcutaneous myeloma cell tumors in vivo. The present data suggest that OSSL_325096 exerts anti‐myeloma activity, at least in part through p97/VCP inhibition.

We previously reported that PU.1 is downregulated in the majority of myeloma cell lines and primary myeloma cells of certain myeloma patients, and conditional expression of PU.1 in such myeloma cell lines induced cell cycle arrest and apoptosis. We found downregulation of IRF4 protein in the U266 myeloma cell line following induction of PU.1. Previous studies reported that knockdown of IRF4 in myeloma cell lines induces apoptosis, prompting us to further investigate the role of IRF4 downregulation in PU.1-induced cell cycle arrest and apoptosis in myeloma cells. PU.1 induced downregulation of IRF4 at the protein level, cell cycle arrest and apoptosis in six myeloma cell lines. Chromatin immunoprecipitation (ChIP) revealed that PU.1 directly binds to the IRF4 promoter, whereas a reporter assay showed that PU.1 may suppress IRF4 promoter activity. Stable expression of IRF4 in myeloma cells expressing PU.1 partially rescued the cells from apoptosis induced by PU.1. As it was reported that IRF4 directly binds to the IRF7 promoter and downregulates its expression in activated B cell-like subtype of diffuse large B cell lymphoma cells, we performed ChIP assays and found that IRF4 directly binds the IRF7 promoter in myeloma cells. It is known that IRF7 positively upregulates interferon-β (IFNβ) and induces apoptosis in many cell types. Binding of IRF4 to the IRF7 promoter decreased following PU.1 induction, accompanied by downregulation of IRF4 protein expression. Knockdown of IRF7 protected PU.1-expressing myeloma cells from apoptosis. Furthermore, IFNβ, which is a downstream target of IRF7, was upregulated in myeloma cells along with IRF7 after PU.1 induction. Finally, we evaluated the mRNA expression levels of PU.1, IRF4 and IRF7 in primary myeloma cells from patients and found that PU.1 and IRF7 were strongly downregulated in contrast to the high expression levels of IRF4. These data strongly suggest that PU.1-induced apoptosis in myeloma cells is associated with IRF4 downregulation and subsequent IRF7 upregulation.

Objective: To evaluate the impact of early mobilization after pediatric liver transplantation in the PICU. Design: A 70-month retrospective before-after study. Setting: Medical and surgical PICU with 20 beds at a tertiary children’s hospital. Patients: Seventy-five patients 2–18 years old who underwent liver transplantation and could walk before surgery. Intervention: We meticulously planned and implemented an early mobilization intervention, a multifaceted framework for early mobilization practice in the PICU focusing on a multidisciplinary team approach. Measurements and Main Results: There was a significant increase in the proportion of patients who received physical therapy in the PICU (66% vs 100%; p < 0.001), especially within the first 48 hours after transplantation (9% vs 78%; p < 0.001). Furthermore, the time spent for physical therapy per eligible patient and per eligible PICU day increased (8.1 min [interquartile range, 0–10.6 min] vs 17.4 min [13.2–26.6 min]; p < 0.001). Compared with patients in the pre-early mobilization period, patients in the post-early mobilization period were able to walk again for more than 50 yards without a rolling walker earlier (28 [16–66] vs 23 [19–31] postoperative days; p = 0.015 by the Gray test), and the length of hospital stay of the post-early mobilization group was shorter than that of the pre-early mobilization group (55 [37–99] vs 40 [31–54] postoperative days; p = 0.012). Conclusions: Through implementation of early mobilization for pediatric patients who underwent liver transplantation, the duration from liver transplantation to regaining the ability to walk again without a rolling walker became shorter. Early mobilization intervention was beneficial for pediatric patients who underwent liver transplantation and could walk before surgery.