Improving Proteomic Identification Using Narrow Isolation Windows with Zeno SWATH Data-Independent Acquisition.

Data-independent acquisition (DIA) techniques such as sequential window acquisition of all theoretical mass spectra (SWATH) acquisition have emerged as the preferred strategies for proteomic analyses. Our study optimized the SWATH-DIA method using a narrow isolation window placement approach, improving its proteomic performance. We optimized the acquisition parameter combinations of narrow isolation windows with different widths (1.9 and 2.9 Da) on a ZenoTOF 7600 (Sciex); the acquired data were analyzed using DIA-NN (version 1.8.1). Narrow SWATH (nSWATH) identified 5916 and 7719 protein groups on the digested peptides, corresponding to 400 ng of protein from mouse liver and HEK293T cells, respectively, improving identification by 7.52 and 4.99%, respectively, compared to conventional SWATH. The median coefficient of variation of the quantified values was less than 6%. We further analyzed 200 ng of benchmark samples comprising peptides from known ratios ofEscherichia coli, yeast, and human peptides using nSWATH. Consequently, it achieved accuracy and precision comparable to those of conventional SWATH, identifying an average of 95,456 precursors and 9342 protein groups across three benchmark samples, representing 12.6 and 9.63% improved identification compared to conventional SWATH. The nSWATH method improved identification at various loading amounts of benchmark samples, identifying 40.7% more protein groups at 25 ng. These results demonstrate the improved performance of nSWATH, contributing to the acquisition of deeper proteomic data from complex biological samples.

[A Case of Resection of T2N0 Colon Cancer with Synchronous Solitary Lung Metastasis].

A 73-year-old man was referred to our hospital for anemia. He underwent a colonoscopy; a 15-mm Ip polyp and a 30- mm type 1 lesion were found in the sigmoid colon. Pathological examination results indicated a well-differentiated adenocarcinoma. Thoracic computed tomography(CT)revealed a mass lesion 12 mm in diameter in the left lung lobe. The patient underwent a laparoscopic sigmoidectomy and D3 lymph node dissection and was discharged in a good condition. He then underwent a diagnostic-therapeutic segmental pulmonary resection for the pulmonary mass. Postoperative pathological findings indicated pT1b(SM), ly0, v0 and pT2(MP), ly1, v1, pN0 for the 2 lesions of the colon. The pulmonary mass was diagnosed as a metastatic adenocarcinoma based on immunostaining examination(CK7: negative, CK20: positive, TTF-1: negative, and CDX-2: positive). The patient is currently under follow-up as an outpatient without recurrence.

Oncologic outcomes after laparoscopic versus open multivisceral resection for local advanced colorectal cancer: A meta-analysis.

Laparoscopic (lap) colectomies for advanced colorectal cancer (CRC) often require resection of other organs. We systematically reviewed currently available literature on lap multi-visceral resection for CRC, with regard to short- and long-term oncological outcomes, and compared them with open procedures. We performed a systematic literature search in MEDLINE, EMBASE, Google Scholar and PubMed from inception to November 30, 2020. The aim of this study was to synthesize short-term and oncological outcomes associated with laparoscopic versus open surgery. Pooled proportions and risk ratios (RRs) were calculated using an inverse variance method. We included six observational cohort studies published between 2012 and 2020 (lap procedures: n = 262; open procedures: n = 273). Collectively, they indicated that postoperative complications were significantly more common after open surgeries than lap surgeries (RR: 0.53; 95% confidence interval [CI]: 0.39-0.72; P < 0.00001), but the two approaches did not significantly differ in positive resection margins (RR: 0.75; 95% CI: 0.38-1.50; P = 0.42), local recurrence (RR: 0.66; 95% CI: 0.28-1.62; P = 0.37), or (based on two evaluable studies) 5-year OS (RR: 0.70; 95% CI: 0.46-1.04; P = 0.08) or 5-year DFS (RR: 0.86; 95% CI: 0.67-1.11) for T4b disease. In conclusion, laparoscopic and open multi-visceral resections for advanced CRC have comparable oncologic outcomes. Although a randomized study would be ideal for further research, no such studies are currently available.

A case of sigmoid colon volvulus with transanal ileus tube placement and Sharon's operation performed safely.

Although endoscopic repair is often performed for sigmoid colon volvulus without intestinal necrosis, surgery is the common choice of treatment due to recurrence. With no established treatment, Hartmann's operation or sigmoid colon resection is often performed. We report a case of a 65-year-old man with transanal ileus tube placement before surgery for sigmoid colon volvulus to prevent recurrence and achieve intestinal decompression followed by Sharon's operation to achieve one-stage anastomosis. The patient showed good postoperative course, with no recurrence 3 months after surgery. This report discusses the usefulness of the transanal ileus tube and Sharon's operation for sigmoid colon volvulus without intestinal necrosis along with a review of the literature.

Human Hepatic Transporter Signature Peptides for Quantitative Targeted Absolute Proteomics: Selection, Digestion Efficiency, and Peptide Stability.

PURPOSE: Quantitative targeted absolute proteomics (QTAP) quantifies proteins by measuring the signature peptides produced from target proteins by trypsin digestion. The selection of signature peptides is critical for reliable peptide quantification. The purpose of this study was to comprehensively assess the digestion efficiency and stability of tryptic peptides and to identify optimal signature peptides for human hepatic transporters and membrane marker proteins. METHODS: The plasma membrane fraction of the human liver was digested at different time points and the peptides were comprehensively quantified using quantitative proteomics. Transporters and membrane markers were quantified using the signature peptides by QTAP. RESULTS: Tryptic peptides were classified into clusters with low digestion efficiency, low stability, and high digestion efficiency and stability. Using the cluster information, we found that a proline residue next to the digestion site or the peptide position in or close to the transmembrane domains lowers digestion efficiency. A peptide containing cysteine at the N-terminus or arginine-glycine lowers peptide stability. Based on this information and the time course of peptide quantification, optimal signature peptides were identified for human hepatic transporters and membrane markers. The quantification of transporters with multiple signature peptides yielded consistent absolute values with less than 30% of coefficient variants in human liver microsomes and homogenates. CONCLUSIONS: The signature peptides selected in the present study enabled the reliable quantification of human hepatic transporters. The QTAP protocol using these optimal signature peptides provides quantitative data on hepatic transporters usable for integrated pharmacokinetic studies.

Stereoselective Synthesis of ß-Alkoxy-ß-amido Vinylbenziodoxoles via Iodo(III)etherification of Ynamides.

A trans-iodo(III)etherification reaction of ynamides with benziodoxole triflate and alcohols is reported. Despite the sensitivity of ynamides and enamides toward Brønsted acid, the reaction could be successfully performed under carefully controlled conditions to afford ß-alkoxy-ß-amido vinylbenziodoxoles in moderate to good yields. The products could be subjected to a sequence of cross-coupling via C-I(III) bond cleavage and electrophilic halogenation of the resulting α-alkoxyenamides, allowing for the preparation of densely functionalized esters.

Water Droplet-in-Oil Digestion Method for Single-Cell Proteomics.

Recent advances in single-cell proteomics highlight the promise of sensitive analyses in limited cell populations. However, technical challenges remain for sample recovery, throughput, and versatility. Here, we first report a water droplet-in-oil digestion (WinO) method based on carboxyl-coated beads and phase transfer surfactants for proteomic analysis using limited sample amounts. This method was developed to minimize the contact area between the sample solution and the container to reduce the loss of proteins and peptides by adsorption. This method increased protein and peptide recovery 10-fold. The proteome profiles obtained from 100 cells using the WinO method highly correlated with those from 10,000 cells using the in-solution digestion method. We successfully applied the WinO method to single-cell proteomics and quantified 462 proteins. Using the WinO method, samples can be easily prepared in a multi-well plate, making it a widely applicable and suitable method for single-cell proteomics.

Targeted proteomics for cancer biomarker verification and validation.

Targeted proteomics is a method that measures the amount of target proteins via liquid chromatography-tandem mass spectrometry and is used to verify and validate the candidate cancer biomarker proteins. Compared with antibody-based quantification methods such as ELISA, targeted proteomics enables rapid method development, simultaneous measurement of multiple proteins, and high-specificity detection of modifications. Moreover, by spiking the internal standard peptide, targeted proteomics detects the absolute amounts of marker proteins, which is essential for determining the cut-off values for diagnosis and thus for multi-institutional validation. With these unique features, targeted proteomics can seamlessly transfer cancer biomarker candidate proteins from the discovery phase to the verification and validation phases, thereby resulting in an accelerated cancer biomarker pipeline. Furthermore, understanding the basic principles, advantages, and disadvantages is necessary to effectively utilize targeted proteomics in cancer biomarker pipelines. This review aimed to introduce the technical principles of targeted proteomics for cancer biomarker verification and validation.

Knockdown of Podocalyxin Post-Transcriptionally Induces the Expression and Activity of ABCB1/MDR1 in Human Brain Microvascular Endothelial Cells.

Podocalyxin (PODXL) is a highly sialylated transmembrane protein that is expressed on the luminal membrane of brain microvascular endothelial cells. To clarify the role of PODXL in the blood-brain barrier (BBB), the present study aimed to investigate the effect of PODXL-knockdown on protein expression, especially the expression of ABCB1/MDR1, in human microvascular endothelial cells (hCMEC/D3). By quantitative proteomics, gene ontology enrichment with differentially expressed proteins showed that PODXL-knockdown influenced the immune response and intracellular trafficking. Among transporters, the protein expression of ABCB1/MDR1 and ABCG2/BCRP was significantly elevated by approximately 2-fold in the PODXL-knockdown cells. In the knockdown cells, the efflux activity of ABCB1/MDR1 was significantly increased, while its mRNA expression was not significantly different from that of the control cells. As receptors and tight junction proteins, levels of low-density lipoprotein receptor-related protein 1 and occludin were significantly increased, while those of transferrin receptor and claudin-11 were significantly decreased in the knockdown cells. The present results suggest that PODXL functions as a modulator of BBB function, including transport, tight junctions, and immune responses. Furthermore, PODXL post-transcriptionally regulates the protein expression and efflux activity of ABCB1/MDR1 at the BBB, which may affect drug distribution in the brain.

Initial entry via the left upper quadrant with an optical trocar in laparoscopic bariatric surgery.

INTRODUCTION: Laparoscopic bariatric surgery (BS) is not readily performed in Japan. To facilitate safe initial access to the abdominal cavity, we insert an optical viewing trocar at a unique site in the left upper quadrant (LUQ). Herein, we describe the technique, its advantages, and outcomes. MATERIALS AND SURGICAL TECHNIQUE: Briefly, the optical trocar is inserted just below the left subcostal margin, 8 cm from the midline. On insertion, layers of the abdominal wall are visualized on the monitor. Depending on the angle of insertion, five, seven, or eight layers are seen. DISCUSSION: In assessing our initial entry technique, used in 21 obese patients undergoing laparoscopic sleeve gastrectomy, we found median insertion time to be 25 seconds. There were no related complications. In nearly all (20/21) patients, the abdominal wall was visualized as seven layers: subcutaneous fat, anterior rectus sheath, rectus abdominis muscle, posterior rectus sheath, transverse abdominis muscle, transversalis fascia, and peritoneum. Understanding the layers of the abdominal wall visualized during optical trocar insertion in the LUQ will provide for safe and rapid initial entry in patients undergoing laparoscopic BS and can further the widespread acceptance of laparoscopic BS.

Diazapentabenzocorannulenium: A Hydrophilic/Biophilic Cationic Buckybowl.

Polycyclic aromatic molecules are promising functional materials for a wide range of applications, especially in organic electronics. However, their largely hydrophobic nature has impeded further applications. As such, imparting high solubility/hydrophilicity to polycyclic aromatic molecules leads to a breakthrough in this research field. Herein, we report the synthesis of diazapentabenzocorannulenium, a cationic nitrogen-embedded buckybowl bearing a central imidazolium core, by a bottom-up strategy from polycyclic aromatic azomethine ylide. X-ray crystallography analyses have revealed a bowl-shaped molecular structure that is capable of forming charge-segregated one-dimensional columns by bowl-in-bowl packing. In addition to its fluorescence capabilities and high dispersibility in water, the molecule was found to selectively localize in the mitochondria of various tumor cells, showing potential as viable mitochondria-selective fluorescent probes.

Effect of Insulin Receptor-Knockdown on the Expression Levels of Blood-Brain Barrier Functional Proteins in Human Brain Microvascular Endothelial Cells.

PURPOSE: The insulin receptor (INSR) mediates insulin signaling to modulate cellular functions. Although INSR is expressed at the blood-brain barrier (BBB), its role in the modulation of BBB function is poorly understood. Therefore, in this study, we aimed to analyze the effect of INSR knockdown on the expression levels of functional proteins at the BBB. METHODS: We established the INSR-knockdown cell line (shINSR) using human cerebral microvascular endothelial cells (hCMEC/D3). The cellular proteome was analyzed using quantitative proteomics. RESULTS: INSR mRNA and protein expressions were decreased in shINSR cells. The suppression of INSR-mediated signaling in shINSR cells was evaluated. The proteins involved in glycolysis and glycogenolysis were suppressed in shINSR cells. As amyloid-ß peptide-related proteins, the expressions of presenilin-1 was increased, and those of the insulin-degrading enzyme and neprilysin were decreased. The expressions of BBB transporters, including the ABCB1/MDR1, ABCG2/BCRP, and SLCO2A1/OATP2A1 were significantly decreased by more than 50% in shINSR cells. The efflux activity of ABCB1/MDR1 was also suppressed. The expressions of the low-density lipoprotein receptor-related protein 1 were significantly increased, and those of the transferrin receptor were significantly decreased in shINSR cells. The expression of claudin-5 was also suppressed in shINSR cells. CONCLUSIONS: The present study suggests that INSR-mediated signaling is involved in the regulation of functional protein expression at the BBB and contributes to the maintenance of BBB function. Changes in the expressions of amyloid-ß peptide-related proteins may contribute to the development of cerebral amyloid angiopathy via the suppression of INSR-mediated signaling.

Regio- and Stereoselective Synthesis of Enol Carboxylate, Phosphate, and Sulfonate Esters via Iodo(III)functionalization of Alkynes.

ß-Iodo(III)enol carboxylates, phosphates, and tosylates can be efficiently synthesized through regio- and stereoselective iodo(III)functionalization of alkynes. The combination of chlorobenziodoxole and silver salt has proven to generate a versatile cationic iodine(III) electrophile to activate alkynes and engage various carboxylic acids, triethyl phosphate, and p-toluenesulfonic acid as nucleophiles. The ß-iodo(III)enol esters serve as starting materials for the synthesis of multisubstituted alkenes through sequential cross-coupling of the C-I(III) and C-O bonds.

Ritter-type iodo(iii)amidation of unactivated alkynes for the stereoselective synthesis of multisubstituted enamides.

The Ritter reaction, Brønsted- or Lewis acid-mediated amidation of alkene or alcohol with nitrile via a carbocation, represents a classical method for the synthesis of tertiary amides. Although analogous reactions through a vinyl cation or a species alike may offer a route to enamide, an important synthetic building block as well as a common functionality in bioactive compounds, such transformations remain largely elusive. Herein, we report a Ritter-type trans-difunctionalization of alkynes with a trivalent iodine electrophile and nitrile, which affords ß-iodanyl enamides in moderate to good yields. Mediated by benziodoxole triflate (BXT), the reaction proves applicable to a variety of internal alkynes as well as to various alkyl- and arylnitriles. The benziodoxole group in the product serves as a versatile handle for further transformations, thus allowing for the preparation of various tri- and tetrasubstituted enamides that are not readily accessible by other means.

A Trial Protocol to Investigate the Incidence of Postoperative Bowel Obstruction after Laparoscopic Colorectal Cancer Surgery Using an Absorbable Adhesion Barrier Material (INTERCEEDⓇ) (Balsam CEED Study): A Prospective, Multicenter, Observational Study.

BACKGROUND: Some studies have reported that adhesion prevention barriers (APBs) reduce adhesion after abdominal surgery; however, evidence showing that APBs reduce the incidence of postoperative small bowel obstruction (SBO), one of the most serious complications after abdominal surgery, is little. One concern is that APBs are usually applied only under the midline incision, although adhesion can occur at any place in the peritoneum where an incision is made during surgery. INTERCEEDⓇ is an APB that reportedly prevents postoperative SBO after surgery. This study aims to assess the clinical utility of INTERCEEDⓇ for the prevention of SBO after laparoscopic colorectal cancer surgery and determine whether the application site of INTERCEEDⓇ affects the incidence of SBO. METHODS/DESIGN: This study is a prospective, multicenter, observational study conducted in Japan. The primary end point is the incidence of postoperative SBO 2 years after laparoscopic colorectal cancer surgery. The secondary end points include whether the site of the application of INTERCEEDⓇ affects the incidence of SBO. Each surgeon selects one of the following three procedures: 1) INTERCEEDⓇ is placed only under the midline incision; 2) INTERCEEDⓇ is placed at the site of bowel mobilization and/or lymph node dissection, but not under the midline incision; and 3) INTERCEEDⓇ is placed at both sites. DISCUSSION: This is the first study to assess whether the placement of APBs affects the incidence of SBO. The study results may lead to a subsequent randomized study.

Iron-catalysed enantioselective carbometalation of azabicycloalkenes.

The first enantioselective carbometalation reaction of azabicycloalkenes has been achieved by iron catalysis to in situ form optically active organozinc intermediates, which are amenable to further synthetic elaborations. The observed chiral induction, along with the DFT and XAS analyses, reveals the direct coordination of the chiral phosphine ligand to the iron centre during the carbon-carbon and carbon-metal bond forming step. This new class of iron-catalysed asymmetric reaction will contribute to the synthesis and production of bioactive molecules.