Two cases of hepatocellular carcinoma successfully treated by carbon ion radiotherapy after atezolizumab plus bevacizumab treatment.

We herein report two cases of huge hepatocellular carcinoma (HCC) that were successfully treated by carbon ion radiotherapy after atezolizumab plus bevacizumab treatment. Case 1, an 84-year-old man, was diagnosed with HCC (maximum diameter: 11 cm) with portal invasion and presented HCC rupture. After obtaining hemostasis with transcatheter embolization, three cycles of atezolizumab-bevacizumab therapy were administered, and marked shrinkage of the HCC was confirmed. However, he developed jaundice, liver damage and cerebral subcortical hemorrhage. Thus, atezolizumab-bevacizumab therapy was discontinued. Total bilirubin, transaminase levels, and physical activity improved well with prednisolone, an antihypertensive agent, and rehabilitation. Thus, treatment with carbon ion radiotherapy (CIRT) was added, and the treatment effect at 4 months after CIRT was judged as a complete response (CR) according to the modified response evaluation criteria in solid tumors (mRECIST). Case 2, a 68-year-old man, was diagnosed with HCC (maximum diameter: 14 cm). Hepatic resection was difficult because the residual liver volume after treatment would be insufficient. Five cycles of atezolizumab-bevacizumab therapy were performed, and marked shrinkage of the HCC to a maximum diameter of 9 cm was confirmed. The treatment was converted to CIRT, and atezolizumab-bevacizumab therapy resumed one month after CIRT. The treatment effect at 3 months after CIRT was judged as CR according to mRECIST. Although conversion therapy after atezolizumab-bevacizumab therapy, including surgery and radiofrequency ablation, have been reported, CIRT may be a promising new tool for conversion therapy for HCC.

Accumulation of Toxic Advanced Glycation End-Products Induces Cytotoxicity and Inflammation in Hepatocyte-Like Cells Differentiated from Human Induced Pluripotent Stem Cells.

Nonalcoholic steatohepatitis (NASH), the aggressive form of the most common chronic liver disease nonalcoholic fatty liver disease, is characterized by inflammation and damage in the liver. Although hepatocyte injury and cell death have been identified as cardinal pathological features of NASH, its pathogenesis has not yet been elucidated in detail. Immortalized cell lines and primary cultured cells have been used as in vitro models of NASH. However, these cells have several disadvantages, such as specialized characteristics by immortalization or limited growth potential. To overcome these difficulties and develop a strategy to analyze the pathology of NASH, we employed hepatocyte-like cells differentiated from human induced pluripotent stem cells (hiPSC-HLCs) as an in vitro model of NASH to clarify the intracellular effects of glyceraldehyde-derived advanced glycation end-products (AGEs), also named toxic AGEs (TAGE). The viability of hiPSC-HLCs decreased with the accumulation of TAGE in the cells, which was consistent with previous findings on human hepatocellular carcinoma cells and human primary cultured hepatocytes. In addition, the TAGE accumulation up-regulated the expression of inflammation-related genes (interleukin 6, interleukin 8, and monocyte chemoattractant protein-1) in hiPSC-HLCs. These results indicated that the accumulation of TAGE induced hiPSC-HLC cytotoxicity and inflammation, which are features of the pathology of NASH. Therefore, we suggest the use of hiPSC-HLCs as an important strategy for analyses of the pathology of NASH.

Specific chemical modification explores dynamic structure of the NqrB subunit in Na+-pumping NADH-ubiquinone oxidoreductase from Vibrio cholerae.

The Na+-pumping NADH-ubiquinone oxidoreductase (Na+-NQR) is a main ion transporter in many pathogenic bacteria. We previously proposed that N-terminal stretch of the NqrB subunit plays an important role in regulating the ubiquinone reaction at the adjacent NqrA subunit in Vibrio cholerae Na+-NQR. However, since approximately three quarters of the stretch (NqrB-Met1-Pro37) was not modeled in an earlier crystallographic study, its structure and function remain unknown. If we can develop a method that enables pinpoint modification of this stretch by functional chemicals (such as spin probes), it could lead to new ways to investigate the unsettled issues. As the first step to this end, we undertook to specifically attach an alkyne group to a lysine located in the stretch via protein-ligand affinity-driven substitution using synthetic ligands NAS-K1 and NAS-K2. The alkyne, once attached, can serve as an "anchor" for connecting functional chemicals via convenient click chemistry. After a short incubation of isolated Na+-NQR with these ligands, alkyne was predominantly incorporated into NqrB. Proteomic analyses in combination with mutagenesis of predicted target lysines revealed that alkyne attaches to NqrB-Lys22 located at the nonmodeled region of the stretch. This study not only achieved the specific modification initially aimed for but also provided valuable information about positioning of the nonmodeled region. For example, the fact that hydrophobic NAS-Ks come into contact with NqrB-Lys22 suggests that the nonmodeled region may orient toward the membrane phase rather than protruding into cytoplasmic medium. This conformation may be essential for regulating the ubiquinone reaction in the adjacent NqrA.

Inhibitors of a Na+-pumping NADH-ubiquinone oxidoreductase play multiple roles to block enzyme function.

The Na+-pumping NADH-ubiquinone (UQ) oxidoreductase (Na+-NQR) is present in the respiratory chain of many pathogenic bacteria and is thought to be a promising antibiotic target. Whereas many details of Na+-NQR structure and function are known, the mechanisms of action of potent inhibitors is not well-understood; elucidating the mechanisms would not only advance drug design strategies but might also provide insights on a terminal electron transfer from riboflavin to UQ. To this end, we performed photoaffinity labeling experiments using photoreactive derivatives of two known inhibitors, aurachin and korormicin, on isolated Vibrio cholerae Na+-NQR. The inhibitors labeled the cytoplasmic surface domain of the NqrB subunit including a protruding N-terminal stretch, which may be critical to regulate the UQ reaction in the adjacent NqrA subunit. The labeling was blocked by short-chain UQs such as ubiquinone-2. The photolabile group (2-aryl-5-carboxytetrazole (ACT)) of these inhibitors reacts with nucleophilic amino acids, so we tested mutations of nucleophilic residues in the labeled region of NqrB, such as Asp49 and Asp52 (to Ala), and observed moderate decreases in labeling yields, suggesting that these residues are involved in the interaction with ACT. We conclude that the inhibitors interfere with the UQ reaction in two ways: the first is blocking structural rearrangements at the cytoplasmic interface between NqrA and NqrB, and the second is the direct obstruction of UQ binding at this interfacial area. Unusual competitive behavior between the photoreactive inhibitors and various competitors corroborates our previous proposition that there may be two inhibitor binding sites in Na+-NQR.

Cholecystectomy in a heifer.

A 10-month-old female Japanese black heifer presenting with sudden loss of appetite was diagnosed with extreme extension of the gallbladder. Laparotomy reaching from the right part of the 10th rib to the right flank showed an extended gallbladder greater than 50 cm in diameter. Cholecystectomy was performed as follows: 1) complete removal of the gallbladder distally from the base; 2) flushing via a catheter inserted into the common bile duct; and 3) covering of the hole opened in the common bile duct with a double-suturing method using the mucous membrane and muscular layers of the remaining gallbladder structures. Serum levels of total bilirubin gradually decreased from 7.5 mg/dl preoperatively to 4.7 mg/dl, 1.6 mg/dl and 0.6 mg/dl at 3, 8 and 34 days postoperatively, respectively. The heifer showed 1 month of clinical improvements, grew normally and finally became pregnant. To the best of our knowledge, this represents the first clinical report to describe cholecystectomy in cattle.

Nasal tissue-derived hamartoma in the maxillary gingiva of a calf.

BACKGROUND: All of oral hamartomas has been previously found in mandibular gingiva in younger calves, and were histologically diagnosed as a vascular hamartoma. This is the first case report describing a calf with a mass in the maxillary gingiva that was histologically diagnosed as a nasal tissue-derived hamartoma. CASE PRESENTATION: A 13-day-old male Holstein calf presented with a horn-like mass in the left rostral maxillary gingiva. Surgical removal revealed that the mass had a dual structure composed of cartilaginous and soft tissues and extended deeply toward the nasal cavity. Excised tissues mainly consisted of two types of mature cells without mitotic figures and atypia: 1) the cartilage-like structures consisted of an island and a meandering massive focus of mature cartilaginous tissues, and 2) tubular structures consisting of stratified ciliated mucosal columnar cells with gland-like structures and aggregated goblet cells. The mass was diagnosed as a nasal tissue-derived hamartoma because these two types of structures were histologically identical to nasal structures. The present case had no recurrence at 1 year after surgery. CONCLUSIONS: To our knowledge, this is the first description of the calf with nasal tissue-derived hamartoma in the maxillary gingiva.