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Rationale and Objectives: To identify if body composition, assessed with preoperative CT-based visceral fat ratio quantification as well as tumor metabolic gene expression, predicts sex-dependent overall survival (OS) in patients with pancreatic ductal adenocarcinoma (PDAC)., Materials and Methods: This was a retrospective analysis of preoperative CT in 98 male and 107 female patients with PDAC. Relative visceral fat (rVFA; visceral fat normalized to total fat) was measured automatically using software and corrected manually. Median and optimized rVFA thresholds were determined according to published methods. Kaplan Meier and log-rank tests were used to estimate OS. Multivariate models were developed to identify interactions between sex, rVFA, and OS. Unsupervised gene expression analysis of PDAC tumors from The Cancer Genome Atlas (TCGA) was performed to identify metabolic pathways with similar survival patterns to rVFA., Results: Optimized preoperative rVFA threshold of 38.9% predicted significantly different OS in females with a median OS of 15 months (above threshold) vs 24 months (below threshold; p = 0.004). No significant threshold was identified in males. This female-specific significance was independent of age, stage, and presence of chronic pancreatitis (p = 0.02). Tumor gene expression analysis identified female-specific stratification from a five-gene signature of glutathione S-transferases. This was observed for PDAC as well as clear cell renal carcinoma and glioblastoma., Conclusion: CT-based assessments of visceral fat can predict pancreatic cancer OS in females. Glutathione S-transferase expression in tumors predicts female-specific OS in a similar fashion., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Joseph Ippolito reports the analysis software license was provided by Vital Images Inc., (Copyright © 2024 The Association of University Radiologists. Published by Elsevier Inc. All rights reserved.)

Background: Functional gastrointestinal disorders (FGIDs) are common early in life. The prevalence of FGIDs varies among countries but is unknown in Vietnam. The aim of this study is to assess the prevalence of FGIDs in healthy Vietnamese infants and young children., Methods: This was a cross-sectional, observational study involving healthy infants and young children between 0 - 48 months old in Hanoi, Vietnam. A representative total of 1511 subjects completed the validated questionnaire on paediatric FGIDs. Rome IV criteria were used to define FGIDs., Results: For Vietnamese infants (0-6 months) and young children (7-48 months), the prevalence of having at least one FGID was 10.0% and only 0.6% was having more than one FGID. Infantile regurgitation (9.3%) was the most prevalent FGID among infants 0-6 months of age while all other FGIDs had a low prevalence between 0-2.5%. For young children between 7 - 48 months old, functional constipation was the most common disorder at the rate of 5.6%. Association analysis revealed that the risk of infant regurgitation was significantly lower among infants with exclusively breastfeeding at 2 - 3 months and 3 - 4 months, formula initiation at 0 - 1 months, and higher paternal education level. The prevalence of functional constipation was significantly higher in male subjects, children in families with annual household income between 273,000,000 - 546,999,999 VND (or estimate around 11,800 - 23,800 USD), families with one child only, and formula feeding initiation at 1 - 2 months., Conclusions: The prevalence of FGIDs in Vietnamese infants and young children is relatively low compared to the published literature using Rome IV diagnostic criteria. Infantile regurgitation was the most common FGID in Vietnamese infants while functional constipation was most prevalent among Vietnamese young children., Trial Registration: Netherlands Trial Registry Identifier: NL7286/NTR7495 ., (© 2022. The Author(s).)

Patients who have undergone mastectomy, with or without reconstruction, are not universally screened with mammography or US. Therefore, clinical breast examination by the physician and patient-detected palpable abnormalities are crucial for detecting breast cancer or recurrence. Diagnostic US is the first-line modality for evaluation of postmastectomy palpable masses, with occasional adjunct use of diagnostic mammography for confirming certain benign masses. In the setting of a negative initial imaging evaluation with continued clinical concern, diagnostic MRI may aid in improving sensitivity. Knowledge of the typical multimodality imaging appearances and locations of malignant palpable abnormalities-such as invasive carcinoma recurrence, cancer in residual breast tissue, radiation-induced sarcoma, and metastatic disease-is crucial in diagnosis and treatment of these entities. In addition, familiarity with the range of benign palpable postmastectomy processes-including fat necrosis, fat graft, seroma, granuloma, neuroma, fibrosis, and infection-may help avoid unnecessary biopsies and reassure patients. The authors review common and rare benign and malignant palpable masses in mastectomy patients, describe multimodality diagnostic imaging evaluation of each entity, review radiologic and pathologic correlation, and acquaint the radiologist with management when these findings are encountered. © RSNA, 2021.

This paper proposes recommendations for probiotics in pediatric gastrointestinal diseases in the Asia-Pacific region. Evidence-based recommendations and randomized controlled trials in the region are included. Cultural aspects, health management issues and economic factors were also considered. Final recommendations were approved by utilizing a modified Delphi process and applying the Likert scale in an electronic voting process. Bacillus clausii was recommended as an adjunct treatment with oral rehydration solution for acute viral diarrhea. B. clausii may also be considered for prevention of antibiotic-associated diarrhea, Clostridium difficile -induced diarrhea, and as adjunct treatment of Helicobacter pylori. There is insufficient evidence for recommendations in other conditions. Despite a diversity of epidemiological, socioeconomical and health system conditions, similar recommendations currently apply to most Asia-Pacific countries. Ideally, these need to be validated with local randomized-controlled trials., Competing Interests: Competing interestsAll authors have disclosed any conflicts of interest. The individual declarations are summarized below. MJG is a member of the SEA Advisory Board of Sanofi-Aventis; JDC is a speaker for Sanofi, Pediatrica and United Laboratories (Unilab Medical Education and Development); DK is a speaker for Nestlé, Danone, Sanofi, Abbott and Dr. Reddy’s Laboratories; KRL is a member of the Advisory Board and Key Opinion Leader of Sanofi, guest speaker for live and webinar meetings (international and national), and National Coordinator and Principal Investigator for research studies; SHQ was a member of the International Data Monitoring Committee (IDMC) for dengue vaccine of Sanofi. All authors received an honorarium from Sanofi for consultancy work. The authors did not receive any payment in relation to writing this publication., (© The Author(s) 2020.)

Purpose To determine if sex differences in abdominal visceral fat composition, measured by using computed tomography (CT), and tumor glucose metabolism, measured by gene expression, can help predict outcomes in patients with clear cell renal cell carcinoma (RCC). Materials and Methods This retrospective cohort study included 222 patients with clear cell RCC from The Cancer Imaging Atlas. By using CT, body fat was segmented into subcutaneous fat and visceral fat areas (VFAs) and normalized to total fat to obtain the relative VFA (rVFA) and relative subcutaneous fat area. Multivariate Cox proportional hazard regression models were performed to identify effects of rVFA on sex-specific survival. Expression profiles for 39 glycolytic genes in tumors from these patients were obtained from The Cancer Genome Atlas to determine sex differences in metabolism and compared with rVFA. Key mutations in clear cell RCC were analyzed for association with rVFA and tumor glycolytic profiles. Results Women with rVFA greater than 30.9% had an increased risk of death (hazard ratio, 3.66 [95% confidence interval: 1.64, 8.19]) for women vs 1.13 ([95% confidence interval: 0.58, 2.18] for men, P = .028). Glycolytic gene expression stratified both men and women, and the combination of low rVFA and low glycolysis identified 19 women with excellent overall survival (P < .001). SETD2 and BAP1 mutations were uniquely enriched in female tumors with high glycolysis (P = .036 and .001, respectively). No significant differences were identified in tumor mutations between patients with high and low rVFA. Conclusion Sex differences in visceral fat and tumor glucose metabolism may provide a new risk-stratification system for patients with clear cell RCC. © RSNA, 2018 Online supplemental material is available for this article.

Plant knottins are of therapeutic interest due to their high metabolic stability and inhibitory activity against proteinases involved in human diseases. The only knottin-type proteinase inhibitor against porcine pancreatic elastase was first identified from the squash family in 1989. Here, we report the identification and characterization of a knottin-type human neutrophil elastase inhibitor from Hibiscus sabdariffa of the Malvaceae family. Combining proteomic and transcriptomic methods, we identified a panel of novel cysteine-rich peptides, roseltides (rT1-rT8), which range from 27 to 39 residues with six conserved cysteine residues. The 27-residue roseltide rT1 contains a cysteine spacing and amino acid sequence that is different from the squash knottin-type elastase inhibitor. NMR analysis demonstrated that roseltide rT1 adopts a cystine-knot fold. Transcriptome analyses suggested that roseltides are bioprocessed by asparagine endopeptidases from a three-domain precursor. The cystine-knot structure of roseltide rT1 confers its high resistance against degradation by endopeptidases, 0.2 N HCl, and human serum. Roseltide rT1 was shown to inhibit human neutrophil elastase using enzymatic and pull-down assays. Additionally, roseltide rT1 ameliorates neutrophil elastase-stimulated cAMP accumulation in vitro. Taken together, our findings demonstrate that roseltide rT1 is a novel knottin-type neutrophil elastase inhibitor with therapeutic potential for neutrophil elastase associated diseases.

Herein we report a novel enzymatic bioconjugation method to prepare peptide dendrimers. Under the catalysis of a newly discovered peptide ligase, butelase 1, peptide dendrimers of di-, tetra-, and octabranches were successfully synthesized using thiodepsipeptides as acyl donors for ligation with lysyl dendrimeric scaffolds. The efficient assembly of the highly clustered dendrimeric structure highlighted the versatility of butelase 1. We also showed that our synthetic antibacterial peptide dendrimers containing an RLYR motif are highly potent and broadly active against antibiotic-resistant strains.

Macrocyclic compounds have received increasing attention in recent years. With their large surface area, they hold promise for inhibiting protein-protein interactions, a chemical space that was thought to be undruggable. Although many chemical methods have been developed for peptide macrocyclization, enzymatic methods have emerged as a promising new economical approach. Thus far, most enzymes have been shown to act on l-peptides; their ability to cyclize d-amino-acid-containing peptides has rarely been documented. Herein we show that macrocycles consisting of d-amino acids, except for the Asn residue at the ligating site, were efficiently synthesized by butelase 1, an Asn/Asp-specific ligase. Furthermore, by using a peptide-library approach, we show that butelase 1 tolerates most of the d-amino acid residues at the P1'' and P2'' positions., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Enzymes that catalyze efficient macrocyclization or site-specific ligation of peptides and proteins can enable tools for drug design and protein engineering. Here we describe a protocol to use butelase 1, a recently discovered peptide ligase, for high-efficiency cyclization and ligation of peptides and proteins ranging in size from 10 to >200 residues. Butelase 1 is the fastest known ligase and is found in pods of the common medicinal plant Clitoria ternatea (also known as butterfly pea). It has a very simple C-terminal-specific recognition motif that requires Asn/Asp (Asx) at the P1 position and a dipeptide His-Val at the P1' and P2' positions. Substrates for butelase-mediated ligation can be prepared by standard Fmoc (9-fluorenylmethyloxycarbonyl) chemistry or recombinant expression with the minimal addition of this tripeptide Asn-His-Val motif at the C terminus. Butelase 1 achieves cyclizations that are 20,000 times faster than those of sortase A, a commonly used enzyme for backbone cyclization. Unlike sortase A, butelase is traceless, and it can be used for the total synthesis of naturally occurring peptides and proteins. Furthermore, butelase 1 is also useful for intermolecular ligations and synthesis of peptide or protein thioesters, which are versatile activated intermediates necessary for and compatible with many chemical ligation methods. The protocol describes steps for isolation and purification of butelase 1 from plant extract using a four-step chromatography procedure, which takes ∼3 d. We then describe steps for intramolecular cyclization, intermolecular ligation and butelase-mediated synthesis of protein thioesters. Butelase reactions are generally completed within minutes and often achieve excellent yields.

Circular bacteriocins, ranging from 35 to 70 amino acids, are the largest cyclic peptides produced by lactic acid bacteria to suppress growth of other bacteria. Their end-to-end cyclized backbone that enhances molecular stability is an advantage to survive in pasteurization and cooking processes in food preservation, but becomes a disadvantage and challenge in chemical synthesis. They also contain unusually long and highly hydrophobic segments which pose an additional synthetic challenge. Here we report the total synthesis of the three largest circular bacteriocins, AS-48, uberolysin, and garvicin ML, by an efficient chemoenzymatic strategy. A key feature of our synthetic scheme is the use of an Asn-specific butelase-mediated cyclization of their linear precursors, prepared by microwave stepwise synthesis. Antimicrobial assays showed that the AS-48 linear precursor is inactive at concentrations up to 100 μM, whereas the macrocyclic AS-48 is potently active against pathogenic and drug-resistant bacteria, with minimal inhibitory concentrations in a sub-micromolar range.

Unlabelled: Cyclotides are plant-derived, cyclic miniproteins with three interlocking disulfide bonds that have attracted great interests because of their excellent stability and potential as peptide therapeutics. In this study, we characterize the cyclotides of the medicinal plant Clitoria ternatea (butterfly pea) and investigate their biological activities. Using a combined proteomic and transcriptomic method, we identified 41 novel cyclotide sequences, which we named cliotides, making C. ternatea one of the richest cyclotide-producing plants to date. Selected members of the cationic cliotides display potent antibacterial activity specifically against Gram-negative bacteria with minimal inhibitory concentrations as low as 0.5 μm. Remarkably, they also possess prominent immunostimulating activity. At a concentration of 1 μm, cationic cliotides are capable of augmenting the secretion of various cytokines and chemokines in human monocytes at both resting and lipopolysaccharide-stimulated states. Chemokines such as macrophage inflammatory proteins 1α and 1β, interferon γ-induced protein 10, interleukin 8 and tumor necrosis factor α were among the most upregulated with up to 129-fold increase in secretion level. These findings suggest cyclotides can serve as potential candidates for novel immunomodulating therapeutics., Database: The protein sequences reported in this paper (cT13-cT21) are available in the UniProt Knowledgebase under the accession numbers C0HJS0, C0HJS1, C0HJS2, C0HJS3, C0HJS4, C0HJS5, C0HJS6, C0HJS7 and C0HJS8, respectively. The transcriptome data in this paper are available at the Sequence Read Archive database (NCBI) under accession number SRR1613316. The protein precursors reported in this paper (ctc13, ctc15, ctc17-ctc19, ctc21-ctc53) are available at GenBank under the accession numbers KT732712, KT732713, KT732714, KT732715, KT732716, KT732717, KT732718, KT732719, KT732720, KT732721, KT732722, KT732723, KT732724, KT732725, KT732726, KT732727, KT732728, KT732729, KT732730, KT732731, KT732732, KT732733, KT732734, KT732735, KT732736, KT732737, KT732738, KT732739, KT732740, KT732741, KT732742, KT732743, KT732744, KT732745, KT732746, KT732747, KT732748 and KT732749, respectively., (© 2016 Federation of European Biochemical Societies.)

Cyclotides are plant cyclic cysteine-rich peptides (CRPs). The cyclic nature is reported to be gene-determined with a precursor containing a cyclization-competent domain which contains an essential C-terminal Asn/Asp (Asx) processing signal recognized by a cyclase. Linear forms of cyclotides are rare and are likely uncyclizable because they lack this essential C-terminal Asx signal (uncyclotide). Here we show that in the cyclotide-producing plant Clitoria ternatea, both cyclic and acyclic products, collectively named cliotides, can be bioprocessed from the same cyclization-competent precursor. Using an improved peptidomic strategy coupled with the novel Asx-specific endopeptidase butelase 2 to linearize cliotides at a biosynthetic ligation site for transcriptomic analysis, we characterized 272 cliotides derived from 38 genes. Several types of post-translational modifications of the processed cyclotides were observed, including deamidation, oxidation, hydroxylation, dehydration, glycosylation, methylation, and truncation. Taken together, our results suggest that cyclotide biosynthesis involves 'fuzzy' processing of precursors into both cyclic and linear forms as well as post-translational modifications to achieve molecular diversity, which is a commonly found trait of natural product biosynthesis.

An efficient ligase with exquisite site-specificity is highly desirable for protein modification. Recently, we discovered the fastest known ligase called butelase 1 from Clitoria ternatea for intramolecular cyclization. For intermolecular ligation, butelase 1 requires an excess amount of a substrate to suppress the reverse reaction, a feature similar to other ligases. Herein, we describe the use of thiodepsipeptide substrates with a thiol as a leaving group and an unacceptable nucleophile to render the butelase-mediated ligation reactions irreversible and in high yields. Butelase 1 also accepted depsipeptides as substrates, but unlike a thiodesipeptide, the desipeptide ligation was partially reversible as butelase 1 can tolerate an alcohol group as a poor nucleophile. The thiodesipeptide method was successfully applied in N-terminal labeling of ubiquitin and green fluorescent protein using substrates with or without a biotin group in high yields., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Using a recently discovered peptide ligase, butelase 1, we developed a novel method to access protein thioesters in good yield. We successfully combined it with native chemical ligation and sortase-mediated ligation in tandem for protein C-terminal labeling and dual-terminal labeling to exploit the orthogonality of these three ligation methods.

Macrocyclization is a valuable tool for drug design and protein engineering. Although various methods have been developed to prepare macrocycles, a general and efficient strategy is needed. Here we report a highly efficient method using butelase 1 to macrocyclize peptides and proteins ranging in sizes from 26 to >200 residues. We achieved cyclizations that are 20,000 times faster than sortase A, the most widely used ligase for protein cyclization. The reactions completed within minutes with up to 95% yields.

Cystine knot α-amylase inhibitors belong to a knottin family of peptidyl inhibitors of 30-32 residues and contain two to four prolines. Thus far, only four members of the group of cystine knot α-amylase inhibitors have been characterized. Herein, the discovery and characterization of five cystine knot α-amylase inhibitors, allotides C1-C5 (Ac1-Ac5) (1-5), from the medicinal plant Allamanda cathartica are reported using both proteomic and genomic methods. Proteomic analysis showed that 1-5 are 30 amino acids in length with three or four proline residues. NMR determination of 4 revealed that it has two cis- and one trans-proline residues and adopts two equally populated conformations in solution. Determination of disulfide connectivity of 2 by differential S-reduction and S-alkylation provided clues of its unfolding process. Genomic analysis showed that allotide precursors contain a three-domain arrangement commonly found in plant cystine knot peptides with conserved residues flanking the processing sites of the mature allotide domain. This work expands the number of known cystine knot α-amylase inhibitors and furthers the understanding of both the structural and biological diversity of this type of knottin family.

Indocyanine green (ICG) angiography has been used in the evaluation of flap perfusion but the viability threshold has not been elucidated. In this study, we determined the threshold by comparing perfusion, using ICG imaging (SPY imaging system, LifeCell Corporation), to clinical evidence of nonviability in rat abdominal perforator flaps. Abdominal flaps, based on a single perforator, were elevated and re-inset in Sprague-Dawley rats. ICG imaging and clinical assessments were conducted preoperatively, as well as 0, 24, and 48 hours postoperatively. SPY-Q software allowed standardization of the perforator's perfusion for comparison purposes. A total of 278 random percentage measurements were made from postoperative day 0 giving a mean (SE) percentage perfusion of 26.8% (1.6%) and 59.1% (1.3%), respectively, for necrosis and survival (P<0.05). We demonstrate that ICG angiography can be readily analyzed in a perforator flap environment allowing a determination of the perfusion threshold.

Proteases are ubiquitous in nature, whereas naturally occurring peptide ligases, enzymes catalyzing the reverse reactions of proteases, are rare occurrences. Here we describe the discovery of butelase 1, to our knowledge the first asparagine/aspartate (Asx) peptide ligase to be reported. This highly efficient enzyme was isolated from Clitoria ternatea, a cyclic peptide-producing medicinal plant. Butelase 1 shares 71% sequence identity and the same catalytic triad with legumain proteases but does not hydrolyze the protease substrate of legumain. Instead, butelase 1 cyclizes various peptides of plant and animal origin with yields greater than 95%. With Kcat values of up to 17 s(-1) and catalytic efficiencies as high as 542,000 M(-1) s(-1), butelase 1 is the fastest peptide ligase known. Notably, butelase 1 also displays broad specificity for the N-terminal amino acids of the peptide substrate, thus providing a new tool for C terminus-specific intermolecular peptide ligations.

It has been shown recently that MCs are required for differential regulation of the immune response by granulocytic versus monocytic MDSCs. Granulocytic MDSCs promoted parasite clearance, whereas monocytic MDSCs enhanced tumor progression; both activities were abrogated in MC-deficient mice. Herein, we demonstrate that the lack of MCs also influences MDSC trafficking. Preferential trafficking to the liver was not seen in MC-deficient mice. In addition, evidence that the MC mediator histamine was important in MDSC trafficking and activation is also shown. MDSCs express HR1-3. Blockade of these receptors by HR1 or HR2 antagonists reversed the histamine enhancement of MDSC survival and proliferation observed in cell culture. In addition, histamine differentially influenced Arg1 and iNOS gene expression in MDSCs and greatly enhanced IL-4 and IL-13 message, especially in granulocytic MDSCs. Evidence that histamine influenced activity seen in vitro translated to in vivo when HR1 and HR2 antagonists blocked the effect of MDSCs on parasite expulsion and tumor metastasis. All of these data support the MDSC-mediated promotion of Th2 immunity, leading to the suggestion that allergic-prone individuals would have elevated MDSC levels. This was directly demonstrated by looking at the relative MDSC levels in allergic versus control patients. Monocytic MDSCs trended higher, whereas granulocytic MDSCs were increased significantly in allergic patients. Taken together, our studies indicate that MCs and MC-released histamine are critical for MDSC-mediated immune regulation, and this interaction should be taken into consideration for therapeutic interventions that target MDSCs., (© 2014 Society for Leukocyte Biology.)

The field of reconstructive microsurgery is experiencing tremendous growth, as evidenced by recent advances in face and hand transplantation, lower limb salvage after trauma, and breast reconstruction. Common to all of these procedures is the creation of a nutrient vascular supply by microsurgical anastomosis between a single artery and vein. Complications related to occluded arterial inflow and obstructed venous outflow are not uncommon, and can result in irreversible tissue injury, necrosis, and flap loss. At times, these complications are challenging to clinically determine. Since early intervention with return to the operating room to re-establish arterial inflow or venous outflow is key to flap salvage, the accurate diagnosis of early stage complications is essential. To date, there are no biochemical markers or serum assays that can predict these complications. In this study, we utilized a rat model of flap ischemia in order to identify the transcriptional signatures of venous congestion and arterial ischemia. We found that the critical ischemia time for the superficial inferior epigastric fasciocutaneus flap was four hours and therefore performed detailed analyses at this time point. Histolgical analysis confirmed significant differences between arterial and venous ischemia. The transcriptome of ischemic, congested, and control flap tissues was deciphered by performing Affymetrix microarray analysis and verified by qRT-PCR. Principal component analysis revealed that arterial ischemia and venous congestion were characterized by distinct transcriptomes. Arterial ischemia and venous congestion was characterized by 408 and 1536>2-fold differentially expressed genes, respectively. qRT-PCR was used to identify five candidate genes Prol1, Muc1, Fcnb, Il1b, and Vcsa1 to serve as biomarkers for flap failure in both arterial ischemia and venous congestion. Our data suggests that Prol1 and Vcsa1 may be specific indicators of venous congestion and allow clinicians to both diagnose and successfully treat microvascular complications before irreversible tissue damage and flap loss occurs.

Cyclotides are disulfide-rich macrocyclic peptides that display a wide range of bioactivities and represent an important group of plant defense peptide biologics. A few linear variants of cyclotides have recently been identified. They share a high sequence homology with cyclotides but are biosynthetically unable to cyclize from their precursors. All hitherto reported cyclotides and their acyclic variants were isolated from dicot plants of the Rubiaceae, Violaceae, Cucurbitaceae, and recently the Fabaceae and Solanaceae families. Although several cyclotide-like genes in the Poaceae family were known from the data mining of the National Center for Biotechnology Information (NCBI) nucleotide database, their expression at the protein level has yet to be proven. Here, we report the discovery and characterization of nine novel linear cyclotides, designated as panitides L1-9, from the Panicum laxum of the Poaceae family and provide the first evidence of linear cyclotides at the protein level in a monocot plant. Disulfide mapping of panitide L3 showed that it possesses a cystine knot arrangement similar to cyclotides. Several panitides were shown to be active against Escherichia coli and cytotoxic to HeLa cells. They also displayed a high stability against heat and proteolytic degradation. Oxidative folding of the disulfide-reduced panitide L1 showed that it can fold efficiently into its native form. The presence of linear cyclotides in both dicots and monocots suggests their ancient origin and existence before the divergence of these two groups of flowering plants. Moreover, the Poaceae family contains many important food crops, and our discovery may open up new avenues of research using cyclotides and their acyclic variants in crop protection.

Background: The determinants for acquisition of Helicobacter pylori infection remain incompletely understood. The study aim was to investigate risk factors for recurrence in children in Vietnam during 1 year immediately following successful H. pylori eradication., Materials and Methods: In a prospective longitudinal study, 136 children, 3-15 years of age, were seen every 3 months for a total of four visits. Helicobacter pylori infection status was determined by an antigen-in-stool test (Premier Platinum HpSA PLUS) on samples obtained at each visit. A questionnaire was filled out at the start of the study., Results: After 1 year, 30 children had become H. pylori positive, while 17 were lost to follow-up. Low age was the most prominent independent risk factor for recurrence: adjusted hazard ratio (HR) among children aged 3-4, 5-6, and 7-8 years, relative to those aged 9-15 years, were, respectively, 14.3 [95% CI 3.8-53.7], 5.4 [1.8-16.3] and 2.6 [0.7-10.4]. Surprisingly, female sex tended to be associated with increased risk (adjusted HR among girls relative to boys 2.5 [95% CI 1.1-5.9]). No other factors such as sibship size, birth order, bed sharing, sanitary standards, or factual antibiotic dose per kilo bodyweight in the eradication trial were found to be significant risk factors for re-infection., Conclusions: The main risk factor for recurrence with H. pylori was found to be age, with the youngest children running the greatest risk. The finding lends support to the observation that early childhood may be the main age of acquisition of H. pylori infection and for postponing attempts of eradication in high-prevalence areas unless motivated for medical reasons., (© 2012 Blackwell Publishing Ltd.)

Background: Low Helicobacter pylori eradication rates are common in pediatric trials especially in developing countries. The aim of the study was to investigate the role of antibiotic resistance, drug dosage, and administration frequency on treatment outcome for children in Vietnam., Materials and Methods:  Antibiotics resistance of H. pylori was analyzed by the Etest in 222 pretreatment isolates from children 3-15 years of age who were originally recruited in a randomized trial with two treatment regiments: lansoprazole with amoxicillin and either clarithromycin (LAC) or metronidazole (LAM) in two weight groups with once- or twice-daily administration. The study design was an observational study embedded in a randomized trial., Results:  The overall resistance to clarithromycin, metronidazole, and amoxicillin was 50.9%, 65.3%, and 0.5%, respectively. In LAC, eradication was linked to the strains being susceptible to clarithromycin (78.2% vs 29.3%, p = .0001). Twice-daily dosage of proton-pump inhibitor (PPI) and clarithromycin was more effective for eradication than once-daily dosage for resistant strains (50.0% vs 14.7%, p = .004) and tended to be so also for sensitive strains (87.5% vs 65.2%, p = .051). Exact antibiotic dose per body weight resulted in more eradication for resistant strains (45.3% vs 8.0%, p = .006). These differences were less pronounced for the LAM regimen, with twice-daily PPI versus once daily for resistant strains resulting in 69.2% and 50.0% eradication (p = .096), respectively., Conclusions:  Helicobacter pylori clarithromycin resistance was unexpectedly high in young children in Vietnam. Clarithromycin resistance was an important cause for eradication treatment failure. Twice-daily administration and exact antibiotic dosing resulted in more eradicated infections when the strains were antibiotic resistant, which has implications for the study design in pediatric H. pylori eradication trials., (© 2012 Blackwell Publishing Ltd.)

Cyclotides are heat-stable macrocyclic peptides from plants that display a wide range of biological activities. They can be divided into two subfamilies: Möbius or bracelet, based on the presence or absence of a cis-proline residue in loop 5, respectively. Currently, over 150 cyclotides have been discovered, but only four linear variants of the Möbius subfamily have been hitherto isolated. In this study, we report the discovery of two novel cyclotides, hedyotide B1 and hedyotide B2, from the aerial parts of Hedyotis biflora. Hedyotide B1 has a cyclic cystine knot structure typical of cyclotides. Interestingly, hedyotide B2 possesses a linear backbone and is the first linear representative of the bracelet subfamily. Disulfide mapping of hedyotide B2 by a top-down MS/MS approach showed that it shares the same knotted disulfide arrangement as conventional cyclotides. Its unfolding pathway also showed that the penetrating disulfide bond Cys III-VI is the most stable disulfide linkage. Cloning of the gene encoding hedyotide B2 revealed a nonsense mutation that introduces a premature stop codon at the conserved Asn residue position, which is essential for an end-to-end backbone ligation. Biophysical characterization showed that hedyotide B2 was more susceptible to exopeptidase degradation as compared with hedyotide B1. Hedyotide B2 was also inactive against all four tested bacterial strains, whereas hedyotide B1 was bactericidal to Escherichia coli and Streptococcus salivarius at low micromolar concentration. Our results provide a deeper understanding of the structures, functions, and biosynthetic processing of cyclotides and uncyclotides in plants.

The tropical plant Clitoria ternatea is a member of the Fabaceae family well known for its medicinal values. Heat extraction of C. ternatea revealed that the bioactive fractions contained heat-stable cysteine-rich peptides (CRPs). The CRP family of A1b (Albumin-1 chain b/leginsulins), which is a linear cystine knot CRP, has been shown to present abundantly in the Fabaceae. In contrast, the cyclotide family, which also belongs to the cystine knot CRPs but with a cyclic structure, is commonly found in the Rubiaceae, Violaceae, and Cucurbitaceae families. In this study, we report the discovery of a panel of 15 heat-stable CRPs, of which 12 sequences (cliotide T1-T12) are novel. We show unambiguously that the cliotides are cyclotides and not A1bs, as determined by their sequence homology, disulfide connectivity, and membrane active properties indicated by their antimicrobial activities against Escherichia coli and cytotoxicities to HeLa cells. We also show that cliotides are prevalent in C. ternatea and are found in every plant tissue examined, including flowers, seeds, and nodules. In addition, we demonstrate that their precursors are chimeras, half from cyclotide and the other half from Albumin-1, with the cyclotide domain displacing the A1b domain in the precursor. Their chimeric structures likely originate from either horizontal gene transfer or convergent evolution in plant nuclear genomes, which are exceedingly rare events. Such atypical genetic arrangement also implies a different mechanism of biosynthetic processing of cyclotides in the Fabaceae and provides new understanding of their evolution in plants.

Background: Eradication of Helicobacter pylori infection in children in developing countries needs further investigations upon which to base treatment recommendations. The aim of the study was to compare two 2-week triple therapies in a randomized double-blind trial., Materials and Methods: In order not to exceed recommended dosages, the 238 H. pylori-infected children, aged 3 to 15 years (mean 8.6), were divided in two weight categories receiving at weights 13-22 kg: lansoprazole 15 mg once-daily and amoxicillin 500 mg twice-daily with metronidazole 250 mg twice-daily or clarithromycin 250 mg once-daily; at weights 23-45 kg: lansoprazole 15 mg and amoxicillin 750 mg with metronidazole 500 mg or clarithromycin 250 mg, all administered twice daily. H. pylori status was assessed by culture and a monoclonal-based antigen-in-stool test (Premier Platinum HpSA PLUS) and side effects by structured questionnaires., Results: The overall per-protocol eradication (n = 233) was similar in the two treatment regimens, 62.1% for the metronidazole and 54.7% for the clarithromycin-containing therapy. Eradication rate was higher in children >or= 23 kg (70.9%) than in children < 23 kg (45.7%). In children >or= 23 kg (n = 117) that received twice-daily administration of all drugs, efficacy of the metronidazole and clarithromycin-containing treatments were 69.5% and 72.4%, respectively., Conclusions: The two treatments gave similar eradication rates. Significant differences for both treatments were found by weight, which could be the result of the once-daily proton pump inhibitor and clarithromycin and/or more antibiotic resistant strains in younger children.

Background: Helicobacter pylori infection is difficult to diagnose in children, especially in developing countries where noninvasive methods such as urea breath test are often not available. We evaluate the sensitivity and specificity of a new monoclonal antibody-based antigen-in-stool enzyme immunoassay (Premier Platinum HpSA PLUS) for diagnosis of H. pylori infection in Vietnamese children., Materials and Methods: Sensitivity of the antigen-in-stool test was evaluated in 232 children, 3-15 years of age, who were positive for H. pylori infection by culture from biopsies. For evaluation of the specificity 98 children of similar age with nongastrointestinal conditions and who were negative for H. pylori infection by serologic assays were included with blood and stool samples., Results: Of the 232 culture-positive children, 224 were also positive by Premier Platinum HpSA PLUS. Of the 98 control children, 93 were H. pylori negative also in the stool test. The sensitivity of Premier Platinum HpSA PLUS was thus 96.6% (95% CI 93.3-98.5) and the specificity was 94.9% (95% CI 88.5-98.3)., Conclusions: The findings have demonstrated Premium Platinum HpSA PLUS to be a reliable method for detection of H. pylori infection also in children in our area.

Three men and 2 women with ages ranging from 37 to 70 years, clinically and histologically confirmed solitary, palpable metastatic cancers to the thyroid (SMCT) and preoperative cytologic investigation of their thyroid lesions by fine-needle aspiration (FNA), were reviewed. Four patients were known to have a solid cancer treated by radical surgery 1 to 4 years prior [1 bronchogenic squamous cell carcinoma, 1 parotid adenoid cystic carcinoma, 1 renal cell carcinoma (RCC) and 1 cutaneous melanoma], and 1 patient had no past history of cancer. Direct smears prepared from the patients' thyroid FNAs were fixed in 95% ethanol and stained with the Papanicolaou method. In 3 cases, immunostaining of the aspirated tumor cells with thyroglobulin antibody was performed, and in 1 case an aspiration smear was stained with commercial HMB-45 antibody. A correct cytodiagnosis of metastatic cancer to the thyroid was made in all 5 cases. In 1 patient the thyroid FNA revealed a metastatic RCC that led to the discovery of a clinically occult RCC. All 5 patients died of metastatic disease 27 to 40 months after surgical resection of their SMCTs.

Background: Primary esophageal rhabdomyosarcoma (PER) is a very rare neoplasm with only 15 cases reported in the literature. Of those, only 1 case underwent a preoperative cytologic evaluation. We report a case of PER with diagnosis by imprint cytology., Case: A 55-year-old woman presented with dysphagia of 2 months' duration associated with fatigue and weight loss. Clinical and diagnostic imaging investigations revealed a large, submucosal mass lesion located in the lower part of the esophagus. A bite biopsy of the esophageal mass was performed under esophagoscopy. Two imprint smears were made from the biopsied tissue fragment and stained with the May-Grünwald-Giemsa method. The smears revealed abundant, pleomorphic, malignant cells with basophilic cytoplasm. Some spindle-shaped cancer cells showed intracytoplasmic cross-striations, indicating a pleomorphic rhabdomyosarcoma, as confirmed by histologic and immunohistochemical studies of the biopsied tumor tissue and resected tumor., Conclusion: The presence of pleomorphic malignant cells with intracytoplasmic cross striations is a characteristic feature of pleomorphic rhabdomyosarcoma.

Three histologically confirmed minimal deviation endometrioid adenocarcinomas (MDEA) of the uterine cervix with cytologic evaluation by cervical scraping were reviewed. The smears were cellular and showed tall columnar tumor cells arranged in monolayered sheets with nuclei in palisade at free borders, rosettes, and irregular clusters. Cellular strips with palisading nuclei was noted in one case. The individual tumor cells showed monomorphic, round or oval, hyperchromatic nuclei with chromatin clumping, small nucleoli, and granular, nonvacuolated cytoplasm with cytoplasmic extensions or tails. The smear background showed a variable amount of necrotic debris admixed with polymorphonuclear leukocytes in two cases. The cytologic manifestations of those three cervical MDEAs overlapped, to some extents, with those of a cervical adenocarcinoma in situ and with those of a well-differentiated endometrial adenocarcinoma invading the cervix., (2006 Wiley-Liss, Inc.)

This community-based cross-sectional study in 533 participants from 135 households with multiple generations living in the same household aimed at investigating the relationship between Helicobacter pylori infection in children and the other household members. H. pylori infection in children was found significantly associated with the infection in mothers [OR (95% CI): 2.50 (1.19-5.26)], even after being adjusted for sex, age group and sibling number [adjusted OR (95% CI): 2.47 (1.12-5.47)]. It was also significantly associated with the infection in both parents [adjusted OR (95% CI): 4.14 (1.29-13.23)]. No significant association between H. pylori infection in the father, grandparent(s), uncle or aunt with that in their children was found. Results from the present study showed intra-familial transmission in a multi-generation population and supported the hypothesis of person-to-person transmission of H. pylori infection.

Background: Patients who present with facial pigmentation can be a diagnostic challenge., Objective: The goal of this study was to discuss the diagnosis and management of imipramine-induced facial pigmentation., Methods: We describe a patient with facial pigmentation of 26 years' duration that was associated with imipramine treatment for depression. We discuss light and election microscopic findings and review 11 previously reported cases of imipramine-induced skin pigmentation., Results: Examination showed blue-gray facial pigmentation. Light microscopy showed perivascular pigment granule deposits in the upper dermis that stained positively with Fontana-Masson stain and negatively with Prussian blue stain. Electron microscopy showed electron-dense bodies within histiocytes without clearly identifiable melanin granules, consistent with drug-induced pigmentation. Six weeks after switching to sertraline the patient reported a slight improvement of her cutaneous pigmentation., Conclusion: Imipramine is a rare cause of gray-blue facial pigmentation. Light microscopy consistently shows granular dermal deposits that stain positively with Fontana-Masson stain but negatively with iron stain.

Thyroid nodules (TN) are a common clinical problem. Fine needle aspiration (FNA) of the thyroid now is practiced worldwide and proves to be the most economical and reliable diagnostic procedure to identify TNs that need surgical excision and TNs that can be managed conservatively. The key for the success of thyroid FNA consists of an adequate or representative cell sample and the expertise in thyroid cytology. The FNA cytologic manifestations of TNs may be classified into seven working cytodiagnostic groups consisting of a few heterogenous lesions each to facilitate the differential diagnosis. Recent application of diagnostic molecular techniques to aspirated thyroid cells proved to be useful in separating benign from malignant TNs in several cases of indeterminate lesions.

Our hypotheses were (1) that plain radiography of the cervical spine in the evaluation of low risk patients with minor blunt trauma is accurate and (2) that computed tomography (CT) of the cervical spine in the evaluation of low risk patients is unnecessary. This study evaluates those hypotheses. We prospectively recorded findings of all patients with blunt trauma who underwent imaging over a period of 70 days. Injury status was determined by review of all radiographic studies obtained on each patient. Chart reviews were performed to determine mechanism of injury and neurological status. Patients were divided into three groups: very low risk, low risk, and high risk. Study end point was fracture. The study included 219 patients. The very low risk group had 107 patients, none of whom had fractures found on CT (projected specificity of plain radiography: 100%). The low risk group contained 78 patients who had no fractures seen on plain radiography or CT (specificity of plain radiography and CT: 100%). The high risk group consisted of 34 patients; 15 had fractures demonstrated on CT. Plain radiography missed one fracture out of 15. In low risk patients, plain radiography is an efficient diagnostic exam with a specificity of 100%. In high risk patients, plain radiography is a good adjunctive screening exam in conjunction with CT scan, with a sensitivity of 93.3% and specificity of 95%.

A 62-year-old white woman with an unremarkable past medical history presented with acute cholecystitis. A cholecystectomy was performed, revealing an acute hemorrhagic and chronic cholecystitis associated with cholelithiasis. Two months after the operation, the patient developed a massive hemoperitoneum and died by hypo-volemic shock. At autopsy, an angiosarcoma measuring 5 cm in diameter was found in the liver, at the site of the gallbladder fossa. There were multiple hepatic, splenic, ovarian and peritoneal metastases and a massive hemoperitoneum consisting of 8 L of blood and blood clots. Review of the tissue sections from the patient's gallbladder confirmed the presence of an acute hemorrhagic and chronic cholecystitis and also revealed residual foci of an angiosarcoma. A review of eight previously reported cases of gallbladder angiosarcoma is also presented.