Safety of early pelvic drain removal in colorectal surgery based on drainage quantity.

BACKGROUND: This study aimed to investigate the association between the drainage quantity of pelvic drains and postoperative complications in colorectal surgery. MATERIALS AND METHODS: This retrospective single-center study enrolled 122 colorectal surgery patients between January 2017 and December 2020. After restorative proctectomy or proctocolectomy with gastrointestinal anastomosis, a continuous, low-pressure suction pelvic drain was placed and its contents measured. Removal ensued following the absence of turbidity and a drainage quantity of ≤ 150 mL/day. RESULTS: Seventy-five patients (61.5%) and 47 patients (38.5%) underwent restorative proctectomy and proctocolectomy, respectively. Drainage quantity changes were observed on postoperative day (POD) 3, regardless of the surgical procedure or postoperative complications. The median (interquartile range) number of PODs before drain removal and organ-space surgical site infection (SSI) diagnosis were 3 (3‒5) and 7 (5‒8), respectively. Twenty-one patients developed organ-space SSIs. Drains were left in place in two patients after POD 3 owing to large drainage quantities. Drainage quality changes enabled diagnosis in two patients (1.6%). Four patients responded to therapeutic drains (3.3%). CONCLUSIONS: The drainage quantity of negative-pressure closed suction drains diminishes shortly after surgery, regardless of the postoperative course. It is not an effective diagnostic or therapeutic drain for organ-space SSI. This supports early drain removal based on drainage quantity changes in actual clinical practice. TRIAL REGISTRATION: The study protocol was retrospectively registered and carried out per the Declaration of Helsinki and approved by the Hiroshima University Institutional Review Board (approval number: E-2559).

Cupid and Psyche system for the diagnosis and treatment of advanced cancer.

In advanced cancer patients, malignant cells invade and disseminate within normal cells and develop resistance to therapy with additional genetic mutations, which makes radical cure very difficult. Precision medicine against advanced cancer is hampered by the lack of systems aimed at multiple target molecules within multiple loci. Here, we report the development of a versatile diagnostic and therapeutic system for advanced cancer, named the Cupid and Psyche system. Based on the strong non-covalent interaction of streptavidin and biotin, a low immunogenic mutated streptavidin, Cupid, and a modified artificial biotin, Psyche, have been designed. Cupid can be fused with various single-chain variable fragment antibodies and forms tetramer to recognize cancer cells precisely. Psyche can be conjugated to a wide range of diagnostic and therapeutic agents against malignant cells. The Cupid and Psyche system can be used in pre-targeting therapy as well as photo-immunotherapy effectively in animal models supporting the concept of a system for precision medicine for multiple targets within multiple loci.

Epiregulin Recognition Mechanisms by Anti-epiregulin Antibody 9E5: STRUCTURAL, FUNCTIONAL, AND MOLECULAR DYNAMICS SIMULATION ANALYSES.

Epiregulin (EPR) is a ligand of the epidermal growth factor (EGF) family that upon binding to its epidermal growth factor receptor (EGFR) stimulates proliferative signaling, especially in colon cancer cells. Here, we describe the three-dimensional structure of the EPR antibody (the 9E5(Fab) fragment) in the presence and absence of EPR. Among the six complementarity-determining regions (CDRs), CDR1-3 in the light chain and CDR2 in the heavy chain predominantly recognize EPR. In particular, CDR3 in the heavy chain dramatically moves with cis-trans isomerization of Pro(103). A molecular dynamics simulation and mutational analyses revealed that Arg(40) in EPR is a key residue for the specific binding of 9E5 IgG. From isothermal titration calorimetry analysis, the dissociation constant was determined to be 6.5 nm. Surface plasmon resonance analysis revealed that the dissociation rate of 9E5 IgG is extremely slow. The superimposed structure of 9E5(Fab)·EPR on the known complex structure of EGF·EGFR showed that the 9E5(Fab) paratope overlaps with Domains I and III on the EGFR, which reveals that the 9E5(Fab)·EPR complex could not bind to the EGFR. The 9E5 antibody will also be useful in medicine as a neutralizing antibody specific for colon cancer.

Structure-based design and synthesis of a bivalent iminobiotin analog showing strong affinity toward a low immunogenic streptavidin mutant.

The streptavidin/biotin interaction has been widely used as a useful tool in research fields. For application to a pre-targeting system, we previously developed a streptavidin mutant that binds to an iminobiotin analog while abolishing affinity for natural biocytin. Here, we design a bivalent iminobiotin analog that shows 1000-fold higher affinity than before, and determine its crystal structure complexed with the mutant protein.

Structural basis for the different stability and activity between the Cdk5 complexes with p35 and p39 activators.

Cyclin-dependent kinase 5 (Cdk5) is a brain-specific membrane-bound protein kinase that is activated by binding to the p35 or p39 activator. Previous studies have focused on p35-Cdk5, and little is known regarding p39-Cdk5. The lack of functional understanding of p39-Cdk5 is due, in part, to the labile property of p39-Cdk5, which dissociates and loses kinase activity in nonionic detergent conditions. Here we investigated the structural basis for the instability of p39-Cdk5. p39 and p35 contain N-terminal p10 regions and C-terminal Cdk5 activation domains (AD). Although p35 and p39 show higher homology in the C-terminal AD than the N-terminal region, the difference in stability is derived from the C-terminal AD. Based on the crystal structures of the p25 (p35 C-terminal region including AD)-Cdk5 complex, we simulated the three-dimensional structure of the p39 AD-Cdk5 complex and found differences in the hydrogen bond network between Cdk5 and its activators. Three amino acids of p35, Asp-259, Asn-266, and Ser-270, which are involved in hydrogen bond formation with Cdk5, are changed to Gln, Gln, and Pro in p39. Because these three amino acids in p39 do not participate in hydrogen bond formation, we predicted that the number of hydrogen bonds between p39 and Cdk5 was reduced compared with p35 and Cdk5. Using substitution mutants, we experimentally validated that the difference in the hydrogen bond network contributes to the different properties between Cdk5 and its activators.

JNK regulates the photic response of the mammalian circadian clock.

The posttranslational regulation of mammalian clock proteins has been assigned a time-keeping function, but seems to have more essential roles. Here we show that c-Jun N-terminal kinase (JNK), identified by inhibitor screening of BMAL1 phosphorylation at Ser 520/Thr 527/Ser 592, confers dynamic regulation on the clock. Knockdown of JNK1 and JNK2 abrogates BMAL1 phosphorylation and lengthens circadian period in fibroblasts. Mice deficient for neuron-specific isoform JNK3 have altered behavioural rhythms, with longer free-running period and compromised phase shifts to light. The locomotor rhythms are insensitive to intensity variance of constant light, deviating from Aschoff's rule. Thus, JNK regulates a core characteristic of the circadian clock by controlling the oscillation speed and the phase in response to light.

Construction and characterization of functional anti-epiregulin humanized monoclonal antibodies.

Growth factors are implicated in several processes essential for cancer progression. Specifically, epidermal growth factor (EGF) family members, including epiregulin (EREG), are important prognostic factors in many epithelial cancers, and treatments targeting these molecules have recently become available. Here, we constructed and expressed humanized anti-EREG antibodies by variable domain resurfacing based on the three-dimensional (3D) structure of the Fv fragment. However, the initial humanized antibody (HM0) had significantly decreased antigen-binding affinity. Molecular modeling results suggested that framework region (FR) residues latently important to antigen binding included residue 49 of the light chain variable region (VL). Back mutation of the VL49 residue (tyrosine to histidine) generated the humanized version HM1, which completely restored the binding affinity of its murine counterpart. Importantly, only one mutation in the framework may be necessary to recover the binding capability of a humanized antibody. Our data support that HM1 exerts potent antibody-dependent cellular cytotoxicity (ADCC). Hence, this antibody may have potential for further development as a candidate therapeutic agent and research tool.

Transgenic overexpression of USP15 in the heart induces cardiac remodeling in mice.

We found a novel protein-protein interaction between ubiquitin-specific protease 15 (USP15) and skeletal muscle LIM protein 1 (SLIM1): USP15 and SLIM1 directly bound under cell-free conditions and co-immunoprecipitated from the lysates of the cells, where they were co-expressed; and USP15 deubiquitinated SLIM1, resulting in the increase of protein levels of SLIM1. Because SLIM1 is strongly implicated in the pathogenesis of myopathies and cardiomyopathies, we generated transgenic (TG) mice with cardiac-specific overexpression of human USP15. Heart weight to body weight ratios and mRNA levels of fetal gene markers in the heart were significantly higher in USP15-TG mice than in wild-type (WT) mice. Also, protein levels of endogenous murine SLIM1 in the heart were significantly higher in USP15-TG mice than in WT mice. Furthermore, the protein of alternatively spliced isoform of SLIM1 was only detected in the heart of USP15-TG mice, and mRNA levels of this isoform were higher as compared to WT mice. These results indicate that USP15 is involved in the regulation of hypertrophic responses in cardiac muscle through transcriptional and post-translational modulation of SLIM1.

A case of multiple metastatic gastric cancer with primary lesion vanished after administrating nivolumab, and the effect remains even after discontinuance of therapy.

Nivolumab is one of the immune checkpoint inhibitors available for chemotherapy-resistant gastric cancer. There have been few reports of confirmed prominent shrinkage of the primary tumor and some reports of prolonged antitumor effect after discontinuance of the drug, but it is not universal. A 67-year-old male was admitted to our hospital and diagnosed with metastatic gastric cancer that had spread to the bilateral lobe of the liver, distant lymph nodes, and peritoneum. He received five courses of S-1 plus oxaliplatin, followed by three courses of ramucirumab plus paclitaxel leading to disease progression. Then, the patient was administered nivolumab as third-line therapy. Tumor size was markedly reduced after three courses, esophagogastroduodenoscopy (EGD) revealed scar formation on the lower gastric corpus after seven courses, and biopsy specimen showed no malignancy. When a slight lower limb muscle weakness manifested, possibly an immune-related adverse event (irAE) after 15 courses, we stopped administration of nivolumab. The patient has survived for 26 months since his first visit, and elimination of the primary tumor and ascites with noted shrinkage of liver and lymph node metastases have followed for more than 10 months since discontinuance of nivolumab.

A long-term survival case of unresectable gastric cancer with multidisciplinary therapy including immunotherapy and abscopal effect.

The prognosis of unresectable gastric cancer remains poor. Multidisciplinary treatment of unresectable gastric cancer is, therefore, thought to be essential for improving patients' outcomes. Here, we report a successful case of multidisciplinary therapy for unresectable gastric cancer. The patient was a 69-year-old woman who was diagnosed with type 2 gastric cancer with remote lymph node metastases and peritoneal dissemination. Although shrinkage of the primary lesion and remote lymph nodes were observed following chemotherapy, we performed distal gastrectomy to deal with continuous bleeding from the primary lesion. Combination therapy with radiation and chemotherapy was effective for multiple metastases in both subclavian lymph nodes and metachronous multiple axillary lymph nodes. Nivolumab combined with radiation therapy also induced regression of remote lymph node metastases, peritoneal dissemination, and adrenal metastasis. Abscopal effects, i.e., shrinkage of the non-irradiated lesions, were also observed. Thus far, the patient has been able to maintain a good quality of life while receiving continued nivolumab therapy. Multidisciplinary therapy including immunotherapy and abscopal effect may improve the quality of life and contribute to long-term survival of patients with unresectable gastric cancer.

Prediction of the mechanism of action of omuralide (clasto-lactacystin beta-lactone) on human cathepsin A based on a structural model of the yeast proteasome beta5/PRE2-subunit/omuralide complex.

Cathepsin A (CathA) is a lysosomal serine carboxypeptidase that exhibits homology and structural similarity to the yeast and wheat serine carboxypeptidases (CPY and CPW) belonging to the alpha/beta-hydrolase fold family. Human CathA (hCathA) and CPW have been demonstrated to be inhibited by a proteasome (threonine protease) inhibitor, lactacystin, and its active derivative, omuralide (clasto-lactacystin beta-lactone), as well as chymostatin. A hCathA/omuralide complex model constructed on the basis of the X-ray crystal structures of the CPW/chymostatin complex and the yeast proteasome beta-subunit (beta5/PRE2)/omuralide one predicted that the conformation of omuralide in the active-site cleft of proteasome beta5/PRE2 should be very similar to that of chymostatin at the S1 catalytic subsites in the hCathA- and CPW-complexes. The relative positions of the glycine residues, i.e., Gly57 in hCathA, Gly53 in CPW, and Gly47 in beta5/PRE2, present in the oxyanion hole of each enzyme were also highly conserved. These results suggest that omuralide might inhibit hCathA and CPW at the S1 subsite in their active-site clefts through direct binding to the active serine residue.

Arginine residue 155 in the second intracellular loop plays a critical role in rat melanin-concentrating hormone receptor 1 activation.

Melanin-concentrating hormone (MCH) receptor 1 (MCH1R) is a class A G protein-coupled receptor. The MCH system has been linked to a variety of physiological functions, including the regulation of feeding and energy metabolism. We recently reported the importance of a dibasic motif in the membrane-proximal C-terminal region for MCH1R function. Here we reveal that an Arg residue in intracellular loop 2 of MCH1R plays a critical role in receptor function. We analyzed the roles of two distinct motifs, BBXXB and BXBB (in which B is a basic residue and X is a nonbasic residue), located in the three intracellular loops of MCH1R. Triple-substitution mutants of intracellular loops 1 and 3 could still activate calcium mobilization, albeit with lower efficacy or potency. However, mutations in intracellular loop 2 led to a complete loss of induction of signal transduction without changing the high affinity constant (Kd) value. By analyzing a series of single-substitution mutants, a point mutation of Arg155 in intracellular loop 2 was found to be responsible for the signaling pathway elicited by MCH. In addition, substitution at positions corresponding to Arg155 in human MCH receptor 2 and rat somatostatin receptor 2 also markedly abolished their ligand-induced signaling capacities, indicating that this Arg is a recognition determinant in several G protein-coupled receptors.

Three dimensional structural studies of alpha-N-acetylgalactosaminidase (alpha-NAGA) in alpha-NAGA deficiency (Kanzaki disease): different gene mutations cause peculiar structural changes in alpha-NAGAs resulting in different substrate specificities and clinical phenotypes.

BACKGROUND: Kanzaki disease (OMIM#104170) is attributable to a deficiency in alpha-N-acetylgalactosaminidase (alpha-NAGA; E.C.3.2.1.49), which hydrolyzes GalNAcalpha1-O-Ser/Thr. Missense mutations, R329W or R329Q were identified in two Japanese Kanzaki patients. Although they are on the same codon, the clinical manifestation was more severe in R329W because an amino acid substitution led to protein instability resulting in structural change, which is greater in R329W than in R329Q. OBJECTIVE: To examine whether the different clinical phenotypes are attributable to the two mutations. METHODS: Plasma alpha-NAGA activity and urinary excreted glycopeptides were measured and three-dimensional models of human alpha-NAGA and its complexes with GalNAcalpha1-O-Ser and GalNAcalpha1-O-Thr were constructed by homology modeling. RESULTS: Residual enzyme activity was significantly higher in the R329Q- than the R329W mutant (0.022+/-0.005 versus 0.005+/-0.001 nmol/h/ml: p<0.05); the urinary ratios of GalNAcalpha1-O-Ser:GalNAcalpha1-O-Thr were 2:10 and 8:10, respectively. GalNAcalpha1-O-Ser/Thr fit tightly in a narrow space of the active site pocket of alpha-NAGA. GalNAcalpha1-O-Thr requires a larger space to associate with alpha-NAGA because of the side chain (CH3) of the threonine residue. CONCLUSION: Our findings suggest that the association of alpha-NAGA with its substrates is strongly affected by the amino acid substitution at R329 and that the association with GalNAcalpha1-O-Thr is more highly susceptible to structural changes. The residual mutant enzyme in R329W could not associate with GalNAcalpha1-O-Thr and GalNAcalpha1-O-Ser. However, the residual mutant enzyme in R329Q catalyzed GalNAcalpha1-O-Ser to some extent. Therefore, the urinary ratio of GalNAcalpha1-O-Ser:GalNAcalpha1-O-Thr was lower and the clinical phenotype was milder in the R329Q mutation. Structural analysis revealed biochemical and phenotypic differences in these Kanzaki patients with the R329Q and R329W mutation.

Viewing the proteome from oligopeptides and prediction of protein function.

Our research activity of making the lexicon of relatively short oligopeptides has been one of the first steps to view the world of proteome from the perspective of oligopeptides. We propose a new method for the prediction of protein function, especially GeneOntology terms (GO terms), based on statistical characteristics of oligopeptides as an application of the lexicon. In the lexicon, a known function of a protein is inherited to its oligopeptides, and the correspondence between oligopeptides and the function is calculated in the whole proteins. In our method, unknown functions of proteins are predicted by means of the correspondence automatically. We measured the prediction performance using the 28,520 whole human proteins registered in RefSeq for several GO terms by recall-precision graphs. The GO terms include 'membrane', 'nucleus', 'ATP binding', 'hydorolase activity', 'GTP binding', 'intracellular signaling cascade' and 'ubiquitin cycle'. In most cases, it scores 70% recall with 80% precision. The prediction for ATP binding and GTP binding results in quite high performance: it scores 80% recall with 80% precision. Even in the worst case (ubiquitin cycle), it scores 62.6% recall with 80% precision. These results suggest that the proposed method is quite efficient for predicting GO terms.

Design and synthesis of biotin analogues reversibly binding with streptavidin.

Two new biotin analogues, biotin carbonate 5 and biotin carbamate 6, have been synthesized. These molecules were designed to reversibly bind with streptavidin by replacing the hydrogen-bond donor NH group(s) of biotin's cyclic urea moiety with oxygen. Biotin carbonate 5 was synthesized from L-arabinose (7), which furnishes the desired stereochemistry at the 3,4-cis-dihydroxy groups, in 11% overall yield (over 10 steps). Synthesis of biotin carbamate 6 was accomplished from L-cysteine-derived chiral aldehyde 33 in 11% overall yield (over 7 steps). Surface plasmon resonance analysis of water-soluble biotin carbonate analogue 46 and biotin carbamate analogue 47 revealed that KD values of these compounds for binding to streptavidin were 6.7×10(-6) M and 1.7×10(-10) M, respectively. These values were remarkably greater than that of biotin (KD =10(-15) M), and thus indicate the importance of the nitrogen atoms for the strong binding between biotin and streptavidin.

Crystal structure of streptavidin mutant with low immunogenicity.

We previously created a low-immunogenic core streptavidin mutant No. 314 (LISA-314) by replacing six amino-acid residues for use as a delivery tool for an antibody multistep pre-targeting process (Yumura et al., Protein Sci., 22, 213-221, 2013). Here, we performed high-resolution X-ray structural analyses of LISA-314 and wild-type streptavidin to investigate the effect of substitutions on the protein function and the three-dimensional structure. LISA-314 forms a tetramer in the same manner as wild-type streptavidin. The binding mode of d-biotin in LISA-314 is also completely identical to that in wild-type streptavidin, and conformational changes were observed mostly at the side chains of substituted sites. Any large conformational changes corresponding to the reduction of B factors around the substituted sites were not observed. These results demonstrated the LISA-314 acquired low immunogenicity without losing structural properties of original wild-type streptavidin.

Structure-based design of a streptavidin mutant specific for an artificial biotin analogue.

For a multistep pre-targeting method using antibodies, a streptavidin mutant with low immunogenicity, termed low immunogenic streptavidin mutant No. 314 (LISA-314), was produced previously as a drug delivery tool. However, endogenous biotins (BTNs) with high affinity (Kd < 10(-10) M) for the binding pocket of LISA-314 prevents access of exogenous BTN-labelled anticancer drugs. In this study, we improve the binding pocket of LISA-314 to abolish its affinity for endogenous BTN species, therefore ensuring that the newly designed LISA-314 binds only artificial BTN analogue. The replacement of three amino acid residues was performed in two steps to develop a mutant termed V212, which selectively binds to 6-(5-((3aS,4S,6aR)-2-iminohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanoic acid (iminobiotin long tail, IMNtail). Surface plasmon resonance results showed that V212 has a Kd value of 5.9 × 10(-7) M towards IMNtail, but no binding affinity for endogenous BTN species. This V212/IMNtail system will be useful as a novel delivery tool for anticancer therapy.

The basic residues in the membrane-proximal C-terminal tail of the rat melanin-concentrating hormone receptor 1 are required for receptor function.

Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide that plays a key role in food intake. It acts through two G protein-coupled receptors (GPCRs), MCH1R and MCH2R, of which MCH1R is the primary regulator of food intake. We have previously reported that N-linked glycosylation of the extracellular domain of MCH1R is necessary for cell surface expression and signal transduction. We now report a role for the rat MCH1R C-terminal region. We constructed serial C-terminal truncation mutants and determined the resulting changes in protein expression, cell surface expression, ligand binding, and MCH-stimulated calcium influx. By analyzing two mutants, deltaT317 (deletion of 36 C-terminal amino acids) and deltaR321 (deletion of 32 C-terminal amino acids), we found that the region between Phe(318) and Arg(321)) was responsible for signal transduction. A more detailed analysis was performed with single or multiple residue mutations. Single mutations of Arg(319), Lys(320), or Arg(321) exhibited a decrease in the cell surface expression, whereas mutations of either Arg(319) or Lys(320), but not Arg(321), showed a significant reduction in the calcium influx. Furthermore, simultaneous mutations of Arg(319) and Lys(320) produced a pronounced decrease in the efficacy of calcium influx stimulation compared with single mutations. A computational analysis revealed a dibasic amino acid motif that is conserved among many class 1 GPCRs and may be part of the amphiphilic cytoplasmic helix 8 (an eight-cytoplasmic helix). Our results therefore provide new insights into the role of the putative helix 8 in the regulation of GPCR function.

Graph-Theoretical Analysis of Cleavage Pattern: Graph Developmental System and Its Application to Cleavage Pattern of Ascidian Egg: (graph theory/graph developmental system/cleavage pattern/ascidian egg).

The skeletal structure of the embryo is represented by the graph. In the graph, the cells and the connectivities between the cells are reduced to the nodes and edges, respectively. Along cleavage history, the series of graphs is obtained. In this paper I propose a new graph developmental system(GDS) which develops the series of graphs. And I represent and analyze the cleavage pattern of the ascidian egg by GDS. In order to represent it by GDS, at first, the connectivities between the cells are labeled according to the developments of the connectivities at the next time step, and next the cells are labeled. But there are two ways of labeling the cells, then two types of GDS are defined: (1) to label according to the pattern of the connectivities of their descendant cells after two time steps (G-GDS), (2) to label according to the cell fates (C-GDS). The C-GDS of the ascidian egg produces 16 cleavage patterns at 64-cell stage non-deterministically. If some labels are distinguished at 16-cell stage, the C-GDS becomes deterministic at 64-cell stage. GDS will be useful to simulate morphogenesis by the computer graphics.

Rhombomere formation and hind-brain crest cell migration from prorhombomeric origins in mouse embryos.

Prior to rhombomere development, structures called prorhombomeres appear in the mammalian hindbrain. This study clarifies the developmental relationship between prorhombomeres and their descendent rhombomeres and hindbrain crest cells in mouse embryos by focal dye injections at various levels of prorhombomere A (proRhA), proRhB, and proRhC, as well as at their boundaries. ProRhA gives rise to two rhombomeres, rhombomeres 1 and 2 (r1 and r2), as well as to crest cells that migrate into the first pharyngeal arch, including the trigeminal ganglion. ProRhB develops into r3 and r4 and produces crest cells populating the second arch and acousticofacial ganglion. The anterior portion of proRhC gives rise to r5 and r6 and to crest cells migrating into the third pharyngeal arch and the IXth ganglion; its posterior portion develops into r7 and releases crest cells into the fourth pharyngeal arch region as well as the Xth ganglion. These results suggest that the boundaries between prorhombomeres serve as lineage restrictions for both hind-brain neuroepithelial cells and for segmental origins of crest cell populations in mouse embryos. The Hox code of the mouse head can be schematized in a much simpler way based on this prorhombomeric organization of the hind-brain, suggesting that prorhombomeres primarily underlie mammalian hind-brain segmentation.