JGOG2046: a feasibility study of neoadjuvant chemotherapy followed by debulking surgery for clinically diagnosed FIGO stage IVb endometrial cancer.

BACKGROUND: We evaluated the feasibility of neoadjuvant chemotherapy, followed by debulking surgery, for clinically diagnosed FIGO stage IVb endometrial cancer (protocol number: JGOG2046). METHODS: The experimental treatment consisted of 3 cycles of paclitaxel (180 mg/m2) plus carboplatin (AUC5) followed by debulking surgery, including total abdominal hysterectomy, bilateral salpingo-oophorectomy, and 3 cycles of adjuvant chemotherapy. Patients were considered as eligible if they were pathologically diagnosed as primary endometrial cancer, and had both endometrial tumor and distant metastasis confirmed by imaging examinations. The primary endpoint was the incidence of patients who completed debulking surgery after the neoadjuvant chemotherapy. RESULTS: While 51 patients were enrolled from 23 hospitals, the final study cohort consisted of 49 patients with a mean age of 59.0 years. Although the response ratio of the neoadjuvant chemotherapy was 65.3% (95% CI 50.4-78.3%), 67.3% (95% confidence interval (CI) 52.5-80.1%) underwent debulking surgery after the neoadjuvant chemotherapy and 59.2% (95% CI 45.2-71.8%) completed the protocol treatment including 3 courses of adjuvant chemotherapy. The median disease-free survival time was 9.1 months (95% CI 6.5-11.9), while the median overall survival time was 23.2 months (95% CI 11.9-27.8). A patient with sigmoid colon cancer and another with cervical cancer were included in this study. CONCLUSIONS: Neoadjuvant chemotherapy followed by debulking surgery was a feasible and acceptable treatment for metastatic endometrial cancer. (225 words).

Phosphoproteomic of the acetylcholine pathway enables discovery of the PKC-ß-PIX-Rac1-PAK cascade as a stimulatory signal for aversive learning.

Acetylcholine is a neuromodulator critical for learning and memory. The cholinesterase inhibitor donepezil increases brain acetylcholine levels and improves Alzheimer's disease (AD)-associated learning disabilities. Acetylcholine activates striatal/nucleus accumbens dopamine receptor D2-expressing medium spiny neurons (D2R-MSNs), which regulate aversive learning through muscarinic receptor M1 (M1R). However, how acetylcholine stimulates learning beyond M1Rs remains unresolved. Here, we found that acetylcholine stimulated protein kinase C (PKC) in mouse striatal/nucleus accumbens. Our original kinase-oriented phosphoproteomic analysis revealed 116 PKC substrate candidates, including Rac1 activator ß-PIX. Acetylcholine induced ß-PIX phosphorylation and activation, thereby stimulating Rac1 effector p21-activated kinase (PAK). Aversive stimulus activated the M1R-PKC-PAK pathway in mouse D2R-MSNs. D2R-MSN-specific expression of PAK mutants by the Cre-Flex system regulated dendritic spine structural plasticity and aversive learning. Donepezil induced PAK activation in both accumbal D2R-MSNs and in the CA1 region of the hippocampus and enhanced D2R-MSN-mediated aversive learning. These findings demonstrate that acetylcholine stimulates M1R-PKC-ß-PIX-Rac1-PAK signaling in D2R-MSNs for aversive learning and imply the cascade's therapeutic potential for AD as aversive learning is used to preliminarily screen AD drugs.

Histopathological Finding of Microdamage Accumulation in Atypical Subtrochanteric Femoral Fracture.

Atypical femoral fracture is a low-energy stress fracture in the subtrochanteric region or the femoral shaft and is a complication of the long-term use of bisphosphonates. Histopathological findings of atypical femoral fractures have not been clarified. Herein, we report the case of a 61-year-old woman who fell while walking, which prompted her to visit our facility. She had a 7-year history of alendronate use to treat osteoporosis. A radiograph showed an atypical subtrochanteric femoral fracture, following which she underwent a primary surgery, where an intramedullary femoral nail was used. Implant breakage was discovered 8 weeks after the primary surgery. The patient underwent a revision surgery in which the entry point for the revised intramedullary hole was created to prevent varus position. The lag screw was successfully inserted into the center of the femoral head. Cancellous bone, isolated from the right ilium, was autogenously implanted into the fracture site. Fracture healing was promoted using low-intensity pulse ultrasonography. Callus formation was detected on a radiograph, and full weight-bearing was advised 12 weeks after the revision surgery. The fracture had healed completely at 13 months after the revision surgery. The patient was able to walk without support and could independently perform activities of daily life. Laboratory findings suggested that the concentrations of her bone formation markers were normal, while those of bone resorption markers were elevated. Iliac bone histomorphometry did not reveal severely suppressed bone turnover. In the cortex of fracture site, the lacunar density was markedly lower than the osteocyte density, and microcracks were detected, suggesting impaired osteocyte function and a low potential for fracture healing. This case is notable because it helps to clarify the histopathological findings of atypical femoral fractures.

Atypical femoral fracture associated with delayed union for which the cessation of alendronate and daily administration of teriparatide contributed to fracture healing: histopathological evidence of the enhancement in bone formation parameters.

A female patient with systemic lupus erythematosus developed an atypical femoral fracture of the left femur after 5 years of glucocorticoid and alendronate therapy. We performed intramedullary nailing. However, 1 week later, we performed a revision surgery using a locking plate and an iliac bone graft because of displacement at the fracture site. At this stage, alendronate was discontinued and daily teriparatide was introduced and continued for 24 months. Twenty months after the revision surgery, a re-revision surgery was performed with an iliac bone graft because of breakage of the locking plate and fracture non-union. Fracture healing was eventually obtained 15 months after the re-revision surgery. Biopsies of the ilium before the treatment and 20 months after daily teriparatide treatment were evaluated. The histology revealed that proliferating osteoid and cuboidal osteoblasts were detected around the osteoid tissue after teriparatide treatment. Bone histomorphometry findings showed that bone volume parameters and osteoid parameters prominently increased with the teriparatide treatment, but not bone resorption parameters. Laboratory findings revealed the elevation of bone-specific alkaline phosphatase (24 U/L at 7 months) compared to its pre-teriparatide level (8.1 U/L) and increased bone mineral density of the hip (from -0.2 to 0.0 in T-score). These data indicated that the discontinuation of alendronate and initiation of teriparatide treatment activated bone-forming ability in our patient and may have contributed to fracture healing.

Publisher Correction: Dynamic structural states of ClpB involved in its disaggregation function.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Advanced cadaver-based educational seminar for trauma surgery using saturated salt solution-embalmed cadavers.

AIM: Senior surgeons in Japan who participated in "cadaver-based educational seminar for trauma surgery (CESTS)" subsequently stated their interest in seminars for more difficult procedures. Therefore, we held a 1-day advanced-CESTS with saturated salt solution (SSS)-embalmed cadavers and assessed its effectiveness for surgical skills training (SST). METHODS: Data were collected from three seminars carried out from September 2015 to January 2018, including a 10-point self-assessment of confidence levels (SACL) questionnaire on nine advanced surgical skills, and evaluation of seminar content before, just after, and half a year after the seminar. Participants assessed the suitability of the two embalming methods (formalin solution [FAS] and SSS) for SST, just after the seminar. Statistical analysis resulted in P < 0.0167 comparing SACL results from seminar evaluations at the three time points and P < 0.05 comparing FAS to SSS. RESULTS: Forty-three participants carried out surgical procedures of the lung, liver, abdominal aorta, and pelvis and extremity. The SACL scores increased in all skills between before and just after the seminar, but were decreased by half a year after. However, SACL scores of each skill did not change significantly, except for external fixation for pelvic fracture at just after and half a year after. The SSS-embalmed cadavers were evaluated as being more suitable than FAS-embalmed cadavers for each procedure. CONCLUSIONS: Advanced-CESTS using SSS-embalmed cadavers increased the participants' self-confidence just after the seminar, which was maintained after half a year in each skill, except external fixation for pelvic fracture. Therefore, SSS-embalmed cadavers are useful for SST, particularly for surgical repairs.

The fission yeast Greatwall-Endosulfine pathway is required for proper quiescence/G0 phase entry and maintenance.

Cell proliferation and cellular quiescence/G0 phase must be regulated in response to intra-/extracellular environments, and such regulation is achieved by the orchestration of protein kinases and protein phosphatases. Here, we investigated fission yeast potential orthologs (Cek1, Ppk18 and Ppk31) of the metazoan Greatwall kinase (Gwl), which inhibits type-2A protein phosphatase with B55 subunit (PP2AB55 ) by phosphorylating and activating the PP2AB55 inhibitors, α-endosulfine/ARPP-19 (Ensa/ARPP-19). Gwl and Ensa/ARPP-19 regulate mitosis; however, we found Ppk18, Cek1 and Mug134/Igo1, the counterpart of Ensa/ARPP-19, are not essential for normal mitosis but regulate nitrogen starvation (-N)-induced proper G0 entry and maintenance. Genetic and biochemical analyses indicated that the conserved Gwl site (serine 64) was phosphorylated in the G0 phase in a Ppk18-dependent manner, and the phosphorylated Mug134/Igo1 inhibited PP2AB55 in vitro. The alanine substitution of the serine 64 caused defects in G0 entry and maintenance as well as the mug134/igo1+ deletion. These results indicate that PP2AB55 activity must be regulated properly to establish the G0 phase. Consistently, simultaneous deletion of the B55 gene with mug134/igo1+ partially rescued the Mug134/Igo1 mutant phenotype. We suggest that in fission yeast, PP2AB55 regulation by the Ppk18-Mug134/Igo1 pathway is required for G0 entry and establishment of robust viability during the G0 phase.

Electrostatic interactions between middle domain motif-1 and the AAA1 module of the bacterial ClpB chaperone are essential for protein disaggregation.

ClpB, a bacterial homologue of heat shock protein 104 (Hsp104), can disentangle aggregated proteins with the help of the DnaK, a bacterial Hsp70, and its co-factors. As a member of the expanded superfamily of ATPases associated with diverse cellular activities (AAA+), ClpB forms a hexameric ring structure, with each protomer containing two AAA+ modules, AAA1 and AAA2. A long coiled-coil middle domain (MD) is present in the C-terminal region of the AAA1 and surrounds the main body of the ring. The MD is subdivided into two oppositely directed short coiled-coils, called motif-1 and motif-2. The MD represses the ATPase activity of ClpB, and this repression is reversed by the binding of DnaK to motif-2. To better understand how the MD regulates ClpB activity, here we investigated the roles of motif-1 in ClpB from Thermus thermophilus (TClpB). Using systematic alanine substitution of the conserved charged residues, we identified functionally important residues in motif-1, and using a photoreactive cross-linker and LC-MS/MS analysis, we further explored potential interacting residues. Moreover, we constructed TClpB mutants in which functionally important residues in motif-1 and in other candidate regions were substituted by oppositely charged residues. These analyses revealed that the intra-subunit pair Glu-401-Arg-532 and the inter-subunit pair Asp-404-Arg-180 are functionally important, electrostatically interacting pairs. Considering these structural findings, we conclude that the Glu-401-Arg-532 interaction shifts the equilibrium of the MD conformation to stabilize the activated form and that the Arg-180-Asp-404 interaction contributes to intersubunit signal transduction, essential for ClpB chaperone activities.

Dynamic structural states of ClpB involved in its disaggregation function.

The ATP-dependent bacterial protein disaggregation machine, ClpB belonging to the AAA+ superfamily, refolds toxic protein aggregates into the native state in cooperation with the cognate Hsp70 partner. The ring-shaped hexamers of ClpB unfold and thread its protein substrate through the central pore. However, their function-related structural dynamics has remained elusive. Here we directly visualize ClpB using high-speed atomic force microscopy (HS-AFM) to gain a mechanistic insight into its disaggregation function. The HS-AFM movies demonstrate massive conformational changes of the hexameric ring during ATP hydrolysis, from a round ring to a spiral and even to a pair of twisted half-spirals. HS-AFM observations of Walker-motif mutants unveil crucial roles of ATP binding and hydrolysis in the oligomer formation and structural dynamics. Furthermore, repressed and hyperactive mutations result in significantly different oligomeric forms. These results provide a comprehensive view for the ATP-driven oligomeric-state transitions that enable ClpB to disentangle protein aggregates.

Bone Formation Parameters of the Biopsied Ilium Differ between Subtrochanteric and Diaphyseal Atypical Femoral Fractures in Bisphosphonate-Treated Patients.

Atypical femoral fractures (AFFs) are defined as atraumatic or low-trauma fractures located in the subtrochanteric or diaphyseal sites. Long-term bisphosphonates (BPs) are administered to prevent fragility fractures in patients with primary osteoporosis or collagen diseases who are already taking glucocorticoids (GCs). Long-term BP use is one of the most important risk factors for AFFs. Its pathogenesis is characterized by severely suppressed bone turnover (SSBT), but whether the characteristics of patients are different regarding to location of fracture site remains unknown. In this study, we compared the characteristics and bone histomorphometric findings between subtrochanteric and diaphyseal sites in patients with BP-associated AFFs. Nine women with BP-associated AFFs were recruited, including 3 with systemic lupus erythematosus, 2 with rheumatoid arthritis, 2 with primary osteoporosis, 1 with polymyalgia rheumatica, and 1 with sarcoidosis. Patients were divided into the subtrochanteric group (n = 5; average age, 52 years; BP treatment, 5.9 years) and the diaphyseal group (n = 4; average age, 77 years; BP treatment, 2.6 years). Compared with the diaphyseal group, the subtrochanteric group had significantly higher daily GC doses (average, 10.9 vs. 2.3 mg/day) and significantly lower serum 25-hydroxyvitamin-D levels (17.8 vs. 25.6 ng/mL). Bone histomorphometry of the biopsied iliac bone showed SSBT in 3 cases (subtrochanteric, n = 1; diaphyseal, n = 2). Osteoid volume and trabecular thickness were significantly lower in the subtrochanteric group than in the diaphyseal group. Bone formation was inhibited more severely in subtrochanteric than in the diaphyseal group due to the higher GC doses used.

Fusion protein analysis reveals the precise regulation between Hsp70 and Hsp100 during protein disaggregation.

ClpB, a bacterial Hsp100, is a ring-shaped AAA+ chaperone that can reactivate aggregated proteins in cooperation with DnaK, a bacterial Hsp70, and its co-factors. ClpB subunits comprise two AAA+ modules with an interstitial rod-shaped M-domain. The M-domain regulates ClpB ATPase activity and interacts directly with the DnaK nucleotide-binding domain (NBD). Here, to clarify how these functions contribute to the disaggregation process, we constructed ClpB, DnaK, and aggregated YFP fusion proteins in various combinations. Notably, i) DnaK activates ClpB only when the DnaK substrate-binding domain (SBD) is in the closed conformation, affording high DnaK-peptide affinity; ii) although NBD alone can activate ClpB, SBD is required for disaggregation; and iii) tethering aggregated proteins to the activated ClpB obviates SBD requirements. These results indicate that DnaK activates ClpB only when the SBD tightly holds aggregated proteins adjacent to ClpB for effective disaggregation.

Analysis of the cooperative ATPase cycle of the AAA+ chaperone ClpB from Thermus thermophilus by using ordered heterohexamers with an alternating subunit arrangement.

The ClpB/Hsp104 chaperone solubilizes and reactivates protein aggregates in cooperation with DnaK/Hsp70 and its cofactors. The ClpB/Hsp104 protomer has two AAA+ modules, AAA-1 and AAA-2, and forms a homohexamer. In the hexamer, these modules form a two-tiered ring in which each tier consists of homotypic AAA+ modules. By ATP binding and its hydrolysis at these AAA+ modules, ClpB/Hsp104 exerts the mechanical power required for protein disaggregation. Although ATPase cycle of this chaperone has been studied by several groups, an integrated understanding of this cycle has not been obtained because of the complexity of the mechanism and differences between species. To improve our understanding of the ATPase cycle, we prepared many ordered heterohexamers of ClpB from Thermus thermophilus, in which two subunits having different mutations were cross-linked to each other and arranged alternately and measured their nucleotide binding, ATP hydrolysis, and disaggregation abilities. The results indicated that the ATPase cycle of ClpB proceeded as follows: (i) the 12 AAA+ modules randomly bound ATP, (ii) the binding of four or more ATP to one AAA+ ring was sensed by a conserved Arg residue and converted another AAA+ ring into the ATPase-active form, and (iii) ATP hydrolysis occurred cooperatively in each ring. We also found that cooperative ATP hydrolysis in at least one ring was needed for the disaggregation activity of ClpB.

ClpB chaperone passively threads soluble denatured proteins through its central pore.

ClpB disaggregase forms a ring-shaped hexamer that threads substrate proteins through the central pore using energy from ATP. The ClpB protomer consists of an N-terminal domain, a middle domain, and two AAA+ modules. These two AAA+ modules bind and hydrolyze ATP and construct the core of the hexameric ring. Here, we investigated the roles of the two AAA+ modules in substrate threading. BAP is an engineered ClpB that can bind ClpP proteolytic chamber; substrates threaded by BAP are degraded by ClpP. We combined BAP with conserved motif mutations in two AAA+ modules and measured the steady-state rates of threading of soluble denatured proteins by these mutants over a range of substrate concentrations. By fitting the data to the Michaelis-Menten equation, k(cat) and K(m) values were determined. We found that the kinetic parameters of the substrate threading correlate with the type of mutation introduced rather than the ATPase activity of the mutant. Moreover, some mutants having no or marginal ATPase activity could thread denatured proteins significantly. These results indicate that ClpB can passively thread soluble denatured proteins.

Conformational transition of the lid helix covering the protease active site is essential for the ATP-dependent protease activity of FtsH.

When bound to ADP, ATP-dependent protease FtsH subunits adopt either an "open" or "closed" conformation. In the open state, the protease catalytic site is located in a narrow space covered by a lidlike helix. This space disappears in the closed form because the lid helix bends at Gly448. Here, we replaced Gly448 with various residues that stabilize helices. Most mutants retained low ATPase activity and bound to the substrate protein, but lost protease activity. However, a mutant proline substitution lost both activities. Our study shows that the conformational transition of the lid helix is essential for the function of FtsH.

Orientation of the amino-terminal domain of ClpB affects the disaggregation of the protein.

ClpB/Hsp104 efficiently reactivates protein aggregates in cooperation with the DnaK/Hsp70 system. As a member of the AAA+ protein family (i.e. an expanded superfamily of ATPases associated with diverse cellular activities), ClpB forms a ring-shaped hexamer in an ATP-dependent manner. A protomer of ClpB consists of an N-terminal domain (NTD), an AAA+ module, a middle domain and another AAA+ module. In the crystal structures, the NTDs point to two different directions relative to other domains and are not visible in the single-particle cryo-electron microscopy reconstruction, suggesting that the NTD is highly mobile. In the present study, we generated mutants in which the NTD was anchored to other domain by disulfide cross-linking and compared several aspects of ClpB function between the reduced and oxidized mutants, using the wild-type and NTD-truncated ClpB (ClpBΔN) as references. In their oxidized form, the mutants and wild-type bind casein with a similar affinity, although the affinity of ClpBΔN for casein was significantly low. However, the extent of casein-induced stimulation of ATPase, the rate of substrate threading and the efficiency of protein disaggregation of these mutants were all lower than those of the wild-type but similar to those of ClpBΔN. These results indicate that the NTD supports the substrate binding of ClpB and that its conformational shift assists the threading and disaggregation of substrate proteins.

Roles of conserved arginines in ATP-binding domains of AAA+ chaperone ClpB from Thermus thermophilus.

ClpB, a member of the expanded superfamily of ATPases associated with diverse cellular activities (AAA+), forms a ring-shaped hexamer and cooperates with the DnaK chaperone system to reactivate aggregated proteins in an ATP-dependent manner. The ClpB protomer consists of an N-terminal domain, an AAA+ module (AAA-1), a middle domain, and a second AAA+ module (AAA-2). Each AAA+ module contains highly conserved WalkerA and WalkerB motifs, and two arginines (AAA-1) or one arginine (AAA-2). Here, we investigated the roles of these arginines (Arg322, Arg323, and Arg747) of ClpB from Thermus thermophilus in the ATPase cycle and chaperone function by alanine substitution. These mutations did not affect nucleotide binding, but did inhibit the hydrolysis of the bound ATP and slow the threading of the denatured protein through the central pore of the T. thermophilus ClpB ring, which severely impaired the chaperone functions. Previously, it was demonstrated that ATP binding to the AAA-1 module induced motion of the middle domain and stabilized the ClpB hexamer. However, the arginine mutations of the AAA-1 module destabilized the ClpB hexamer, even though ATP-induced motion of the middle domain was not affected. These results indicated that the three arginines are crucial for ATP hydrolysis and chaperone activity, but not for ATP binding. In addition, the two arginines in AAA-1 and the ATP-induced motion of the middle domain independently contribute to the stabilization of the hexamer.

Temperature-dependent regulation of Thermus thermophilus DnaK/DnaJ chaperones by DafA protein.

DafA, a unique 8-kDa protein found in Thermus thermophilus, assembles the chaperones DnaK and DnaJ to produce a DnaK(3)-DnaJ(3)-DafA(3) complex (KJA complex). Although, it is known that DafA is denatured irreversibly at nonphysiological 89 degrees C and the KJA complex dissociates into fully active DnaK and DnaJ, the function of the KJA complex is not fully understood. In this article, we report that the reversible dissociation of the KJA complex occurs in a temperature-dependent manner even below physiological 75 degrees C and that excess DafA completely inhibits the chaperone activities of the DnaK system. The inhibited activities are not rescued by supplementing DnaK or DnaJ. The results indicate that DafA inhibits the chaperone activities of both DnaK and DnaJ by forming the KJA complex and can act as a thermosensor under both heat stress and optimal growth conditions.

Stability of the two wings of the coiled-coil domain of ClpB chaperone is critical for its disaggregation activity.

The ClpB chaperone forms a hexamer ring and rescues aggregated proteins in co-operation with the DnaK system. Each subunit of ClpB has two nucleotide-binding modules, AAA (ATPase associated with various cellular activities)-1 and AAA-2, and an 85-A (1 A=0.1 nm)-long coiled-coil. The coiled-coil consists of two halves: wing-1, leaning toward AAA-1, and wing-2, leaning away from all the domains. The coiled-coil is stabilized by leucine zipper-like interactions between leucine and isoleucine residues of two amphipathic alpha-helices that twist around each other to form each wing. To destabilize the two wings, we developed a series of mutants by replacing these residues with alanine. As the number of replaced residues increased, the chaperone activity was lost and the hexamer became unstable. The mutants, which had a stable hexameric structure but lost the chaperone activities, were able to exert the threading of soluble denatured proteins through their central pore. The destabilization of wing-1, but not wing-2, resulted in a several-fold stimulation of ATPase activity. These results indicate that stability of both wings of the coiled-coil is critical for full functioning of ClpB, but not for the central-pore threading of substrate proteins, and that wing-1 is involved in the communication between AAA-1 and AAA-2.