Longitudinal phosphoproteomics reveals the PI3K-PAK1 axis as a potential target for recurrent colorectal liver metastases.

The resistance of colorectal cancer liver metastases (CRLMs) to 5-fluorouracil (5-FU) chemotherapy remains a significant global health challenge. We investigated the phosphoproteomic dynamics of serial tissue sections obtained from initial metastases and recurrent tumors collected from 24 patients to address this unmet need for innovative therapeutic strategies for patients with CRLM with a poor prognosis. Our analysis revealed the activation of PAK kinase in patients with CRLM with a poor prognosis.

Quantitation and characterization of serotype 6A activation for pneumococcal conjugate vaccine by cryo-EM and SEC methods.

Pneumococcal conjugate vaccines (PCV) typically consist of capsular polysaccharides from different S. pneumoniae serotypes which are covalently attached to carrier protein. A well-established process to manufacture PCV is through activating polysaccharide by oxidation of vicinal diols to aldehydes, followed by protein conjugation via reductive amination.

Integrative analysis of cancer dependency data and comprehensive phosphoproteomics data revealed the EPHA2-PARD3 axis as a cancer vulnerability in KRAS-mutant colorectal cancer.

Colorectal cancer (CRC), a common malignant tumour of the gastrointestinal tract, is a life-threatening cancer worldwide. Mutations in KRAS and BRAF, the major driver mutation subtypes in CRC, activate the RAS pathway, contribute to tumorigenesis in CRC and are being investigated as potential therapeutic targets. Despite recent advances in clinical trials targeting KRASG12C or RAS downstream signalling molecules for KRAS-mutant CRC, there is a lack of effective therapeutic interventions.

Systematic identification of ALK substrates by integrated phosphoproteome and interactome analysis.

The sensitivity of phosphorylation site identification by mass spectrometry has improved markedly. However, the lack of kinase-substrate relationship (KSR) data hinders the improvement of the range and accuracy of kinase activity prediction. In this study, we aimed to develop a method for acquiring systematic KSR data on anaplastic lymphoma kinase (ALK) using mass spectrometry and to apply this method to the prediction of kinase activity.

Descending necrotizing mediastinitis in a healthy young adult.

A 26-year-old man with right lower mandibular and chest pain, fever, and respiratory distress was urgently transported to our hospital. CT images revealed gas collection and an abscess from the neck to the mediastinum with bilateral pleural effusion. Descending necrotizing mediastinitis (DNM) induced by an odontogenic infection of a right mandibular molar abscess was diagnosed.

Designing antioxidant peptides based on the antioxidant properties of the amino acid side-chains.

Amino acids exert characteristic antioxidant activities depending on the properties of their side residues. The hydrophobic residues were effective against peroxyl radical, while acidic residues and their analogs were effective against peroxynitrite. Peptides containing tyrosine showed different activities against different reactive oxygen species (ROS) and/or reactive nitrogen species (RNS).

Antioxidant activities of a peptide derived from chicken dark meat.

Antioxidant activities against hypochlorite ions and peroxyl radicals of a chicken dark meat hydrolysate digested with pepsin were examined with the myoglobin method based on the structure change of myoglobin due to redox reaction with reactive oxygen species (ROS). A peptide that showed strong antioxidant activity against the peroxyl radical was isolated from the hydrolysate using HPLC equipped with a hydrophobic-interacting column. The sequence of the first five amino acid residues of the peptide was determined as YASGR (Tyr-Ala-Ser-Gly-Arg), and this sequence matched with the amino acid residues 143-147 of chicken β-actin (GenBank: CAA25004.

Improving the in vivo therapeutic index of siRNA polymer conjugates through increasing pH responsiveness.

Polymer based carriers that aid in endosomal escape have proven to be efficacious siRNA delivery agents in vitro and in vivo; however, most suffer from cytotoxicity due in part to a lack of selectivity for endosomal versus cell membrane lysis. For polymer based carriers to move beyond the laboratory and into the clinic, it is critical to find carriers that are not only efficacious, but also have margins that are clinically relevant. In this paper we report three distinct categories of polymer conjugates that improve the selectivity of endosomal membrane lysis by relying on the change in pH associated with endosomal trafficking, including incorporation of low pKa heterocycles, acid cleavable amino side chains, or carboxylic acid pH sensitive charge switches.

Enhanced binding of calmodulin to the ryanodine receptor corrects contractile dysfunction in failing hearts.

Aims: The channel function of the cardiac ryanodine receptor (RyR2) is modulated by calmodulin (CaM). However, the involvement of CaM in aberrant Ca(2+) release in diseased hearts remains unclear. Here, we investigated the pathogenic role of defective CaM binding to the RyR2 in the channel dysfunction associated with heart failure.

Mutation-linked defective interdomain interactions within ryanodine receptor cause aberrant Ca²⁺release leading to catecholaminergic polymorphic ventricular tachycardia.

Background: The molecular mechanism by which catecholaminergic polymorphic ventricular tachycardia is induced by single amino acid mutations within the cardiac ryanodine receptor (RyR2) remains elusive. In the present study, we investigated mutation-induced conformational defects of RyR2 using a knockin mouse model expressing the human catecholaminergic polymorphic ventricular tachycardia-associated RyR2 mutant (S2246L; serine to leucine mutation at the residue 2246).

Methods And Results: All knockin mice we examined produced ventricular tachycardia after exercise on a treadmill.

Aberrant interaction of calmodulin with the ryanodine receptor develops hypertrophy in the neonatal cardiomyocyte.

We have shown previously that the inter-domain interaction between the two domains of RyR (ryanodine receptor), CaMBD [CaM (calmodulin)-binding domain] and CaMLD (CaM-like domain), activates the Ca(2+) channel, and this process is called activation-link formation [Gangopadhyay and Ikemoto (2008) Biochem. J. 411, 415-423].

Effects of conformational peptide probe DP4 on bidirectional signaling between DHPR and RyR1 calcium channels in voltage-clamped skeletal muscle fibers.

In skeletal muscle, excitation-contraction coupling involves the activation of dihydropyridine receptors (DHPR) and type-1 ryanodine receptors (RyR1) to produce depolarization-dependent sarcoplasmic reticulum Ca²⁺ release via orthograde signaling. Another form of DHPR-RyR1 communication is retrograde signaling, in which RyRs modulate the gating of DHPR. DP4 (domain peptide 4), is a peptide corresponding to residues Leu²⁴⁴²-Pro²⁴⁷⁷ of the central domain of the RyR1 that produces RyR1 channel destabilization.

Intracellular translocation of calmodulin and Ca2+/calmodulin-dependent protein kinase II during the development of hypertrophy in neonatal cardiomyocytes.

We have recently shown that stimulation of cultured neonatal cardiomyocytes with endothelin-1 (ET-1) first produces conformational disorder within the ryanodine receptor (RyR2) and diastolic Ca(2+) leak from the sarcoplasmic reticulum (SR), then develops hypertrophy (HT) in the cardiomyocytes (Hamada et al., 2009 [3]). The present paper addresses the following question.

Dynamic, inter-subunit interactions between the N-terminal and central mutation regions of cardiac ryanodine receptor.

Naturally occurring mutations in the cardiac ryanodine receptor (RyR2) have been linked to certain types of cardiac arrhythmias and sudden death. Two mutation hotspots that lie in the N-terminal and central regions of RyR2 are predicted to interact with one another and to form an important channel regulator switch. To monitor the conformational dynamics involving these regions, we generated a fluorescence resonance energy transfer (FRET) pair.

Dissociation of calmodulin from cardiac ryanodine receptor causes aberrant Ca(2+) release in heart failure.

Aims: Calmodulin (CaM) is well known to modulate the channel function of the cardiac ryanodine receptor (RyR2). However, the possible role of CaM on the aberrant Ca(2+) release in diseased hearts remains unclear. In this study, we investigated the state of RyR2-bound CaM and channel dysfunctions in pacing-induced failing hearts.

Defective calmodulin binding to the cardiac ryanodine receptor plays a key role in CPVT-associated channel dysfunction.

Calmodulin (CaM), one of the accessory proteins of the cardiac ryanodine receptor (RyR2), is known to play a significant role in the channel regulation of the RyR2. However, the possible involvement of calmodulin in the pathogenic process of catecholaminergic polymorphic ventricular tachycardia (CPVT) has not been investigated. In this study, we investigated the state of RyR2-bound CaM and channel dysfunctions using a knock-in (KI) mouse model with CPVT-linked RyR2 mutation (R2474S).

Catecholaminergic polymorphic ventricular tachycardia is caused by mutation-linked defective conformational regulation of the ryanodine receptor.

Rationale: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is caused by a single point mutation in a well-defined region of the cardiac type 2 ryanodine receptor (RyR)2. However, the underlying mechanism by which a single mutation in such a large molecule produces drastic effects on channel function remains unresolved.

Objective: Using a knock-in (KI) mouse model with a human CPVT-associated RyR2 mutation (R2474S), we investigated the molecular mechanism by which CPVT is induced by a single point mutation within the RyR2.

Highly efficient asymmetric synthesis of sitagliptin.

A highly efficient synthesis of sitagliptin, a potent and selective DPP-4 inhibitor for the treatment of type 2 diabetes mellitus (T2DM), has been developed. The key dehydrositagliptin intermediate 9 is prepared in three steps in one pot and directly isolated in 82% yield and >99.6 wt % purity.

Dantrolene, a therapeutic agent for malignant hyperthermia, markedly improves the function of failing cardiomyocytes by stabilizing interdomain interactions within the ryanodine receptor.

Objectives: We sought to investigate the effect of dantrolene, a drug generally used to treat malignant hyperthermia, on the Ca2+ release and cardiomyocyte function in failing hearts.

Background: The N-terminal (N: 1-600) and central (C: 2000-2500) domains of the ryanodine receptor (RyR) harbor many mutations associated with malignant hyperthermia in skeletal muscle RyR (RyR1) and polymorphic ventricular tachycardia in cardiac RyR (RyR2). There is strong evidence that interdomain interaction between these regions plays an important role in the mechanism of channel regulation.

Defective regulation of the ryanodine receptor induces hypertrophy in cardiomyocytes.

Recent studies on cardiac hypertrophy animal model suggest that inter-domain interactions within the ryanodine receptor (RyR2) become defective concomitant with the development of hypertrophy (e.g. de-stabilization of the interaction between N-terminal and central domains of RyR2; T.

Alternative splicing of RyR1 alters the efficacy of skeletal EC coupling.

Alternative splicing of ASI residues (Ala(3481)-Gln(3485)) in the skeletal muscle ryanodine receptor (RyR1) is developmentally regulated: the residues are present in adult ASI(+)RyR1, but absent in the juvenile ASI(-)RyR1 which is over-expressed in adult myotonic dystrophy type 1 (DM1). Although this splicing switch may influence RyR1 function in developing muscle and DM1, little is known about the properties of the splice variants. We examined excitation-contraction (EC) coupling and the structure and interactions of the ASI domain (Thr(3471)-Gly(3500)) in the splice variants.

Amyloid-β protein impairs Ca2+ release and contractility in skeletal muscle.

Inclusion body myositis (IBM), the most common muscle disorder in the elderly, is partly characterized by dysregulation of β-amyloid precursor protein (βAPP) expression and abnormal, intracellular accumulation of full-length βAPP and β-amyloid epitopes. The present study examined the effects of β-amyloid accumulation on force generation and Ca(2+) release in skeletal muscle from transgenic mice harboring human βAPP and assessed the consequence of Aβ(1-42) modulation of the ryanodine receptor Ca(2+) release channels (RyRs). β-Amyloid laden muscle produced less peak force and exhibited Ca(2+) transients with smaller amplitude.