Verification of blood flow path reconstruction mechanism in distal sigmoid colon and rectal cancer after high IMA ligation through preoperative and postoperative comparison by manual subtraction CTA.

INTRODUCTION: We aimed to investigate manual subtraction computed tomography angiography (MS-CTA) to further confirm the distribution and classification of LCA (left colic artery) ascending/descending branches, then observe the postoperative blood flow path to illustrate how the above branches evolved to postoperative blood path. MATERIAL AND METHODS: 89 patients with distal sigmoid and rectal cancer were referred in our observation and underwent MS-CTA between June 2020 and March 2022. We classified the distribution of LCA and confirmed whether there exists AMCA (accessory middle colic artery). Then we planned blood flow path based on the classification of LCA branches before operation. High ligation was applied in regular radical surgery. During operation, we carefully protect the bifurcation of ascending and descending LCA. Then we compared the planned blood flow path with the actual postoperative blood flow path to verify the mechanism we proposed previously. RESULTS: Of 89 patients, 82 cases met our criteria, we summarized 6 distribution pattens of LCA ascending and descending branches. These preoperative pattens are consistent with the inspection during operation. The postoperative blood flow path of 6 pattens is evolved from the above adjacent anastomotic branches and is consistent with the planned blood flow path. We also found 2 cases with IMA stenosis and 1 case with SMA stenosis under pathological condition, and their compensatory blood flow path is in accordance with our theory. The rate of the anastomotic leakage in our study group is relatively low (7.3%). CONCLUSION: MS-CTA could confirm the distribution of LCA and AMCA, display accurate postoperative blood reconstruction path after IMA high ligation, and it further verified the mechanism we proposed previously, which is the proximal anastomotic branches forming new blood flow path from high-pressure area to the low-pressure area. This mechanism might be helpful for performing accurate laparoscopic sigmoid and rectal cancer surgery.

Melittin Tryptophan Substitution with a Fluorescent Amino Acid Reveals the Structural Basis of Selective Antitumor Effect and Subcellular Localization in Tumor Cells.

Melittin is a membrane-active peptide with strong anticancer activity against various cancers. Despite decades of research, the role of the singular Trp in the anticancer activity and selectivity of melittin remains poorly understood. Here, we propose a theranostic solution based on the substitution of Trp19 with a noncanonical fluorescent amino acid (DapAMCA). The introduction of DapAMCA residue in melittin stabilized the helical structure of the peptide, as evaluated by circular dichroism spectra and molecular dynamics simulations. In vitro hemolytic and anticancer activity assays revealed that introducing DapAMCA residue in melittin changed its mode of action with the cell membrane, resulting in reduced hemolytic toxicity and an improved the selectivity index (SI), with up to a five-fold increase compared to melittin. In vitro fluorescence imaging of DapAMCA-labeled melittin (MELFL) in cancer cells demonstrated high membrane-penetrating activity, with strong nuclear and nucleolar localization ability. These findings provide implications for novel anticancer therapies based on Trp-substituted designs and nuclear/nucleolar targeted therapy.

Preparation of the luciferase-labeled antibody for improving the detection sensitivity of viral antigen.

BACKGROUND: Viral antigen detection test is the most common method used to detect viruses in the field rapidly. However, due to the low sensitivity, it can only be used as an auxiliary diagnosis method for virus infection. Improving sensitivity is crucial for developing more accurate viral antigen tests. Nano luciferase (Nluc) is a sensitive reporter that has not been used in virus detection. RESULTS: In this study, we produced an intracellularly Nluc labeled detection antibody (Nluc-ch2C5) and evaluated its ability to improve the detection sensitivity of respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens. Compared with the traditional horse-radish peroxidase (HRP) labeled antibody (HRP-ch2C5), Nluc-ch2C5 was 41 times more sensitive for inactivated SARS-CoV-2 virus by sandwich chemiluminescence ELISA. Then we applied Nluc-ch2C5 to establish an automatic magnet chemiluminescence immune assay (AMCA) for the SARS-CoV-2 viral spike protein, the limit of detection was 68 pfu/reaction. The clinical sensitivity and specificity reached 75% (24/32) and 100% (48/48) using 32 PCR-positive and 48 PCR-negative swab samples for clinical evaluation, which is more sensitive than the commercial ELSA kit and colloid gold strip kit. CONCLUSIONS: Here, monoclonal antibody ch2C5 served as a model antibody and the SARS-CoV-2 served as a model pathogen. The Nluc labeled detecting antibody (Nluc-ch2C5) significantly improved the detection sensitivity of SARS-CoV-2 antigen. This labeling principle applies to other viral infections, so this labeling and test format could be expected to play an important role in detecting other virus antigens.

Real-Time Prediction of Mortality, Cardiac Arrest, and Thromboembolic Complications in Hospitalized Patients With COVID-19.

Background: COVID-19 infection carries significant morbidity and mortality. Current risk prediction for complications in COVID-19 is limited, and existing approaches fail to account for the dynamic course of the disease. Objectives: The purpose of this study was to develop and validate the COVID-HEART predictor, a novel continuously updating risk-prediction technology to forecast adverse events in hospitalized patients with COVID-19. Methods: Retrospective registry data from patients with severe acute respiratory syndrome coronavirus 2 infection admitted to 5 hospitals were used to train COVID-HEART to predict all-cause mortality/cardiac arrest (AM/CA) and imaging-confirmed thromboembolic events (TEs) (n = 2,550 and n = 1,854, respectively). To assess COVID-HEART's performance in the face of rapidly changing clinical treatment guidelines, an additional 1,100 and 796 patients, admitted after the completion of development data collection, were used for testing. Leave-hospital-out validation was performed. Results: Over 20 iterations of temporally divided testing, the mean area under the receiver operating characteristic curve were 0.917 (95% confidence interval [CI]: 0.916-0.919) and 0.757 (95% CI: 0.751-0.763) for prediction of AM/CA and TE, respectively. The interquartile ranges of median early warning times were 14 to 21 hours for AM/CA and 12 to 60 hours for TE. The mean area under the receiver operating characteristic curve for the left-out hospitals were 0.956 (95% CI: 0.936-0.976) and 0.781 (95% CI: 0.642-0.919) for prediction of AM/CA and TE, respectively. Conclusions: The continuously updating, fully interpretable COVID-HEART predictor accurately predicts AM/CA and TE within multiple time windows in hospitalized COVID-19 patients. In its current implementation, the predictor can facilitate practical, meaningful changes in patient triage and resource allocation by providing real-time risk scores for these outcomes. The potential utility of the predictor extends to COVID-19 patients after hospitalization and beyond COVID-19.

A new use of ß-Ala-Lys (AMCA) as a transport reporter for PEPT1 and PEPT2 in renal brush border membrane vesicles from the outer cortex and outer medulla.

Integral membrane proteins PEPT1 and PEPT2 are essential for reabsorbing almost all hydrolysed or filtered di- and tripeptides alongside a wide range of peptidomimetic drugs in the kidney. The aim of this study was to investigate the potential use of the fluorophore-conjugated dipeptide ß-Ala-Lys (AMCA) as a biosensor for measuring peptide transport activity in brush border membrane vesicles isolated from the outer cortex (BBMV-OC) and outer medulla (BBMV-OM) (representing PEPT1 and PEPT2 respectively). The vesicles were isolated using a dual magnesium precipitation and centrifugation technique. Intravesicular fluorescence accumulation was measured after incubating extra-vesicular media at pH6.6 and different concentrations of ß-Ala-Lys (AMCA) with vesicles pre-equilibrated at pH7.4. Both BBMV-OC and BMMV-OM showed accumulation of an intravesicular fluorescence signal after 20min incubation. Changing the extra-vesicular pH to 7.4 caused a significant reduction in the ß-Ala-Lys (AMCA) uptake into BBMV-OC at concentrations >100µM. When different concentrations of dipeptide, Gly-Gln was added, there was a significant inhibition of 100µM ß-Ala-Lys (AMCA) uptake into BBMV-OC and BMMV-OM, reaching 69% and 80%, respectively. Kinetic analysis of ß-Ala-Lys (AMCA) at 20min showed that the Km and Vmax were 783.7±115.7µM and 2191.2±133.9ΔF/min/mg for BBMV-OC, while BMMV-OM showed significantly higher affinity, but lower capacity at Km=93.6±21.9µM and Vmax=935.8±50.2ΔF/min/mg. These findings demonstrate the applicability of ß-Ala-Lys (AMCA) as a biosensor to measure the transport activity of the renal-type PEPT1 and PEPT2 in BBMV-OC and BMMV-OM respectively.

AmcA-a putative mitochondrial ornithine transporter supporting fungal siderophore biosynthesis.

Iron is an essential nutrient required for a wide range of cellular processes. The opportunistic fungal pathogen Aspergillus fumigatus employs low-molecular mass iron-specific chelators, termed siderophores, for uptake, storage and intracellular iron distribution, which play a crucial role in the pathogenicity of this fungus. Siderophore biosynthesis (SB) depends on coordination with the supply of its precursor ornithine, produced mitochondrially from glutamate or cytosolically via hydrolysis of arginine. In this study, we demonstrate a role of the putative mitochondrial transporter AmcA (AFUA_8G02760) in SB of A. fumigatus. Consistent with a role in cellular ornithine handling, AmcA-deficiency resulted in decreased cellular ornithine and arginine contents as well as decreased siderophore production on medium containing glutamine as the sole nitrogen source. In support, arginine and ornithine as nitrogen sources did not impact SB due to cytosolic ornithine availability. As revealed by Northern blot analysis, transcript levels of siderophore biosynthetic genes were unresponsive to the cellular ornithine level. In contrast to siderophore production, AmcA deficiency did only mildly decrease the cellular polyamine content, demonstrating cellular prioritization of ornithine use. Nevertheless, AmcA-deficiency increased the susceptibility of A. fumigatus to the polyamine biosynthesis inhibitor eflornithine, most likely due to the decreased ornithine pool. AmcA-deficiency decreased the growth rate particularly on ornithine as the sole nitrogen source during iron starvation and sufficiency, indicating an additional role in the metabolism and fitness of A. fumigatus, possibly in mitochondrial ornithine import. In the Galleria mellonella infection model, AmcA-deficiency did not affect virulence of A. fumigatus, most likely due to the residual siderophore production and arginine availability in this host niche.

Antibodies against glycoprotein 2 are novel markers of intestinal inflammation in patients with an ileal pouch.

BACKGROUND AND AIMS: The Crohn's disease (CD)-specific pancreatic auto-antibodies (PAB), have been recently identified to target glycoprotein 2 (GP2). Pouchitis is an inflammation of the small bowel developing in up to 60% of ulcerative colitis patients undergoing proctocolectomy and ileal pouch anal anastomosis. Occurrence of CD-specific antibodies was reported to be a predictor of pouchitis. We aimed to assess the prevalence of anti-GP2 antibodies (anti-GP2) in the serum and feces of pouch patients and to correlate them with clinical parameters. Furthermore, we examined mucosal expression of the GP2 protein in the pouch. METHODS: Pouch patients were prospectively recruited and checked for clinical, endoscopic, and laboratory markers of inflammation. IgG and IgA anti-GP2 levels in serum and fecal samples were determined using ELISA. GP2 protein was assessed by immunohistochemistry. RESULTS: Anti-GP2 was elevated in both serum and fecal samples of patients with inflamed compared to those with non-inflamed pouches and patients with familial-adenomatous polyposis after surgery (p<0.05, respectively). Moreover, patients with CD-like complications exhibited significantly higher anti-GP2 titers than those without CD-like complications (p≤0.01). High levels of anti-GP2 correlated with more frequent bowel movements per day and with the presence of at least one anti-glycan antibody (p≤0.05). GP2 itself was more abundant in the mucosa of patients with chronic pouchitis. CONCLUSIONS: Anti-GP2 exists in the serum and feces of pouch patients and correlates with pouch inflammation, and presence of other serological markers. Thus, anti-GP2 may contribute to better stratification of pouchitis, more-so when the inflammation exhibits CD-like complications.