Accessing Diverse Pyridine-Based Macrocyclic Peptides by a Two-Site Recognition Pathway.

Macrocyclic peptides are sought-after molecular scaffolds for drug discovery, and new methods to access diverse libraries are of increasing interest. Here, we report the enzymatic synthesis of pyridine-based macrocyclic peptides (pyritides) from linear precursor peptides. Pyritides are a recently described class of ribosomally synthesized and post-translationally modified peptides (RiPPs) and are related to the long-known thiopeptide natural products. RiPP precursors typically contain an N-terminal leader region that is physically engaged by the biosynthetic proteins that catalyze modification of the C-terminal core region of the precursor peptide. We demonstrate that pyritide-forming enzymes recognize both the leader region and a C-terminal tripeptide motif, with each contributing to site-selective substrate modification. Substitutions in the core region were well-tolerated and facilitated the generation of a wide range of pyritide analogues, with variations in macrocycle sequence and size. A combination of the pyritide biosynthetic pathway with azole-forming enzymes was utilized to generate a thiazole-containing pyritide (historically known as a thiopeptide) with no similarity in sequence and macrocycle size to the naturally encoded pyritides. The broad substrate scope of the pyritide biosynthetic enzymes serves as a future platform for macrocyclic peptide lead discovery and optimization.

Genome Mining for New Enzyme Chemistry.

A revolution in the field of biocatalysis has enabled scalable access to compounds of high societal values using enzymes. The construction of biocatalytic routes relies on the reservoir of available enzymatic transformations. A review of uncharacterized proteins predicted from genomic sequencing projects shows that a treasure trove of enzyme chemistry awaits to be uncovered. This Review highlights enzymatic transformations discovered through various genome mining methods and showcases their potential future applications in biocatalysis.

Biosynthesis of macrocyclic peptides with C-terminal ß-amino-α-keto acid groups by three different metalloenzymes.

Advances in genome sequencing and bioinformatics methods have identified a myriad of biosynthetic gene clusters (BGCs) encoding uncharacterized molecules. By mining genomes for BGCs containing a prevalent peptide-binding domain used for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), we uncovered a new class involving modifications installed by a cytochrome P450, a multi-nuclear iron-dependent non-heme oxidative enzyme (MNIO, formerly DUF692), a cobalamin- and radical S-adenosyl-L-methionine-dependent enzyme (B12-rSAM), and a methyltransferase. All enzymes encoded by the BGC were functionally expressed in Burkholderia sp. FERM BP-3421. Structural characterization with 2D-NMR and Marfey's method on the resulting RiPP demonstrated that the P450 enzyme catalyzed the formation of a biaryl C-C crosslink between two Tyr residues with the B12-rSAM generating ß-methyltyrosine. The MNIO transformed a C-terminal Asp residue into aminopyruvic acid while the methyltransferase acted on the ß-carbon of the α-keto acid. Exciton-coupled circular dichroism spectroscopy and microcrystal electron diffraction (MicroED) were used to elucidate the stereochemical configurations of the atropisomer that formed upon biaryl crosslinking. The conserved Cys residue in the precursor peptide was not modified as in all other characterized MNIO-containing BGCs; However, mutational analyses demonstrated that it was essential for the MNIO activity on the C-terminal Asp. To the best of our knowledge, the MNIO featured in this pathway is the first to modify a residue other than Cys. This study underscores the utility of genome mining to discover new macrocyclic RiPPs and that RiPPs remain a significant source of previously undiscovered enzyme chemistry.

Biosynthesis of Macrocyclic Peptides with C-Terminal ß-Amino-α-keto Acid Groups by Three Different Metalloenzymes.

Advances in genome sequencing and bioinformatics methods have identified a myriad of biosynthetic gene clusters (BGCs) encoding uncharacterized molecules. By mining genomes for BGCs containing a prevalent peptide-binding domain used for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), we uncovered a new compound class involving modifications installed by a cytochrome P450, a multinuclear iron-dependent non-heme oxidative enzyme (MNIO, formerly DUF692), a cobalamin- and radical S-adenosyl-l-methionine-dependent enzyme (B12-rSAM), and a methyltransferase. All enzymes were functionally expressed in Burkholderia sp. FERM BP-3421. Structural characterization demonstrated that the P450 enzyme catalyzed the formation of a biaryl C-C cross-link between two Tyr residues with the B12-rSAM generating ß-methyltyrosine. The MNIO transformed a C-terminal Asp residue into aminopyruvic acid, while the methyltransferase acted on the ß-carbon of this α-keto acid. Exciton-coupled circular dichroism spectroscopy and microcrystal electron diffraction (MicroED) were used to elucidate the stereochemical configuration of the atropisomer formed upon biaryl cross-linking. To the best of our knowledge, the MNIO featured in this pathway is the first to modify a residue other than Cys. This study underscores the utility of genome mining to isolate new macrocyclic RiPPs biosynthesized via previously undiscovered enzyme chemistry.

Bioinformatics-guided discovery of biaryl-linked lasso peptides.

Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) that feature an isopeptide bond and a distinct lariat fold. A growing number of secondary modifications have been described that further decorate lasso peptide scaffolds. Using genome mining, we have discovered a pair of lasso peptide biosynthetic gene clusters (BGCs) that include cytochrome P450 genes. Using mass spectrometry, stable isotope incorporation, and extensive 2D-NMR spectrometry, we report the structural characterization of two unique examples of (C-N) biaryl-linked lasso peptides. Nocapeptin A, from Nocardia terpenica, is tailored with a Trp-Tyr crosslink, while longipepetin A, from Longimycelium tulufanense, features a Trp-Trp linkage. Besides the unusual bicyclic frame, a Met of longipepetin A undergoes S-methylation to yield a trivalent sulfonium, a heretofore unprecedented RiPP modification. A bioinformatic survey revealed additional lasso peptide BGCs containing P450 enzymes which await future characterization. Lastly, nocapeptin A bioactivity was assessed against a panel of human and bacterial cell lines with modest growth-suppression activity detected towards Micrococcus luteus.

Bioinformatics-Guided Discovery of Biaryl-Tailored Lasso Peptides.

Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) that feature an isopeptide bond and a distinct lariat fold. A growing number of secondary modifications have been described that further decorate lasso peptide scaffolds. Using genome mining, we have discovered a pair of lasso peptide biosynthetic gene clusters (BGCs) that include cytochrome P450 genes. Here, we report the structural characterization of two unique examples of (C-N) biaryl-containing lasso peptides. Nocapeptin A, from Nocardia terpenica, is tailored with Trp-Tyr crosslink while longipepetin A, from Longimycelium tulufanense, features Trp-Trp linkage. Besides the unusual bicyclic frame, longipepetin A receives an S-methylation by a new Met methyltransferase resulting in unprecedented sulfonium-bearing RiPP. Our bioinformatic survey revealed P450(s) and further maturating enzyme(s)-containing lasso BGCs awaiting future characterization.

A method for non-destructive determination of cocoa bean fermentation levels based on terahertz hyperspectral imaging.

Fermentation of cocoa is a key process to obtain aromatic chocolate products from raw cocoa beans. Hitherto, the levels of fermentation in cocoa are determined using destructive techniques, for example by a cut-test to manually observe the colour inside the beans, or by quantifying ammonia nitrogen (NH3) in the cocoa powder. In this paper, we present the use of Terahertz hyperspectral imaging as a new way to non-destructively analyse and detect fermented cocoa beans. The study analysed two sets of twenty-two cocoa bean samples with different levels of fermentation from two producers in Brazil. A correlation between fermentation conditions and the outcome results of their THz measurements was observed.

Chest wall reconstruction of severe mediastinitis with intercostal artery-based pedicled vertical rectus abdominis muscle flap with oblique-designed skin pedicle.

Recurrent poststernotomy mediastinitis has significant morbidity and mortality. Reconstructive treatment begins with pectoral muscle or omental flaps. When these options are unavailable or inadequate, surgeons resort to internal mammary artery-based vertical rectus abdominis muscle flap. If the internal mammary artery is harvested for coronary artery bypass grafting, surgeons are reluctant to use the muscle for pedicled flap because of the elevated risks. However, recent reports suggest that if enough time passes for intercostal artery collaterals to develop, they would support the viability of the flap. Moreover, recent improvements in defining flap microvasculature and proposed surgical techniques have enabled us to further refine the procedure. Although it appears that the intercostal artery-based pedicled vertical rectus abdominis muscle flap with oblique-designed skin pedicle is safe and effective for chest wall reconstruction, potential for failure remains elevated until sample size accumulates.

Microdeformation of three-dimensional cultured fibroblasts induces gene expression and morphological changes.

BACKGROUND: Vacuum-assisted closure induces microdeformations of the wound surface and accelerates healing of complex wounds; however, a thorough understanding of the biology of cellular mechanotransduction is lacking. We hypothesized that fibroblast shape and function can be altered in an in vitro vacuum-assisted closure device. METHODS: A 3-dimensional fibrin matrix with cultured murine fibroblasts and an intervening polyurethane foam was exposed to 125 mm Hg suction and compared with similar wells without suction. We measured fibroblast proliferation and morphology using fluorescence microscopy and gene expression change using real-time reverse-transcriptase polymerase chain reaction at 24, 48, and 72 hours. RESULTS: Wells exposed to suction induced significant proliferation of fibroblasts and morphologic changes visible by larger, rounder, and notable dendrite-like branching and process extensions. Type 1 collagen alpha 1 (COL1A1), fibroblast growth factor 2 (FGF2, bFGF), and transforming growth factor beta 1 (TGFß1) were all up-regulated after 48 hours of exposure to suction. Smooth muscle actin alpha 2 (Acta2, α-SMA) was up-regulated after 72 hours. CONCLUSIONS: Microdeformations produced by the combination of polyurethane foam and suction are associated with increased fibroblast proliferation and up-regulation of gene expressions in fibroblasts.

Description of a new species of the Charaeacoomani group (Coleoptera: Chrysomelidae: Galerucinae) from Vietnam with a key to species.

BACKGROUND: The genus Charaea Baly is distributed in the eastern Palaearctic, Himalayas, China and adjacent countries of the Oriental Region. Currently, 59 species of the genus Charaea have been recorded. The species of Charaea is characterised with a robust tubular aedeagus that terminates with a more or less distinct apical process with the Charaeacoomani group having an internal sac with long sharp lateral sclerites. Up to now, 13 species of this group have been described in the Oriental Region, four of which are found in Vietnam. NEW INFORMATION: Charaeadinhcuongi sp. nov. is described as a new species, based on specimens collected from Phu Quoc Island in southern Vietnam. Colour photographs of habitus and body details and DNA barcode sequences are presented. An identification key is provided for all Vietnamese species from the Charaeacoomani group.

Use of the parabiotic model in studies of cutaneous wound healing to define the participation of circulating cells.

Previous experimental studies to assess the contribution of blood-borne circulating (BBC) cells to cutaneous wound healing have relied on discontinuous pulsing of labeled BBC elements or bone marrow transplant protocols. Such approaches do not allow the examination of stable BBC cells that have matured in a physiologically normal host. We have used a parabiotic murine model for cutaneous wound healing to evaluate the relative contribution of stable populations of peripheral blood cells expressing the green fluorescent protein (GFP) transgene in otherwise normal animals. Circulating cells (mature and immature) expressing the GFP transgene were easily detected and quantified in wounds of GFP- parabiotic twins during all evaluated stages of the healing response. Using multiple antibody probes, the relative contribution of various subsets of BBC cells could be comparatively assessed. In early wounds, some cells expressing mesenchymal epitopes were documented to be of hematopoietic origin, indicating the utility of this model in assessing cell plasticity in the context of tissue regeneration and repair. Application of this approach enables further investigation into the contribution of peripheral blood in normal and abnormal healing responses.

Subtle Ecological Gradient in the Tropics Triggers High Species-Turnover in a Local Geographical Scale.

Our perception of diversity, including both alpha- and beta-diversity components, depends on spatial scale. Studies of spatial variation of the latter are just starting, with a paucity of research on beta-diversity patterns at smaller scales. Understanding these patterns and the processes shaping the distribution of diversity is critical to describe this diversity, but it is paramount in conservation too. Here, we investigate the diversity and structure of a tropical community of herbivorous beetles at a reduced local scale of some 10 km2, evaluating the effect of a small, gradual ecological change on this structure. We sampled leaf beetles in the Núi Chúa National Park (S Vietnam), studying changes in alpha- and beta-diversity across an elevation gradient up to 500 m, encompassing the ecotone between critically endangered lowland dry deciduous forest and mixed evergreen forest at higher elevations. Leaf beetle diversity was assessed using several molecular tree-based species delimitation approaches (with mtDNA cox1 data), species richness using rarefaction and incidence-based diversity indexes, and beta-diversity was investigated decomposing the contribution of species turnover and nestedness. We documented 155 species in the area explored and species-richness estimates 1.5-2.0x higher. Species diversity was similar in both forest types and changes in alpha-diversity along the elevation gradient showed an expected local increase of diversity in the ecotone. Beta-diversity was high among forest paths (average Sørensen's dissimilarity = 0.694) and, tentatively fixing at 300 m the boundary between otherwise continuous biomes, demonstrated similarly high beta-diversity (Sørensen's dissimilarity = 0.581), with samples clustering according to biome/elevation. Highly relevant considering the local scale of the study, beta-diversity had a high contribution of species replacement among locales (54.8%) and between biomes (79.6%), suggesting environmental heterogeneity as the dominant force shaping diversity at such small scale, directly and indirectly on the plant communities. Protection actions in the Park, especially these addressed at the imperative conservation of dry forest, must ponder the small scale at which processes shape species diversity and community structure for inconspicuous, yet extraordinarily diverse organisms such as the leaf beetles.

Syndrome of inappropriate secretion of antidiuretic hormone in association with pulmonary carcinosarcoma.

A smoking patient presented with altered mental status, left sided weakness, and an opacity on the chest roentgenogram. During hospitalization he developed hyponatremia, which was corrected with hypertonic saline solution, fluid restriction, and demethylchlortetracycline. The clinical presentation and the laboratory findings supported the diagnosis of the syndrome of inappropriate secretion of antidiuretic hormone. This was believed to be associated with the pulmonary carcinosarcoma documented at necropsy.

The sternalis muscle-incidental finding of a rare chest wall muscle variant during keloid excision-chest wall reconstruction.

INTRODUCTION: The sternalis is a rare (strap-like) parasternal muscle that is well known to anatomists, but relatively unknown to clinicians, including surgeons. Familiarity with the muscle is important in avoiding potential confusion when incidentally encountered. When available, the muscle can be harvested for reconstruction of the chest wall or of nearby region. PRESENTATION OF CASE: The patient is a 55-year-old man with significant sternal keloids secondary to a previous history of severe acne. The patient desires removal of the keloids. Hence, a decision was made to excise the keloids, followed by immediate reconstruction with a propeller flap. Intraoperative excision of the keloids and undermining of adjacent subcutaneous tissue revealed chest muscle fibers fitting the description of the sternalis muscle. The patient tolerated the procedure without any complications. DISCUSSION: The sternalis muscle can be confused for a mass on mammography, but confusion is resolved by computed tomography/magnetic resonance imaging. It has unclear embryonic origin-perhaps a remnant of the panniculus carnosus and/or derivative of a primitive ventral-longitudinal muscle sheet that give rise to the sternocleidomastoid and the rectus abdominis muscles. It is uni- or bilateral and has 1 or 2 bellies. It originates in the intraclavicular region and inserts onto the rectus sheath, costal cartilages, lower ribs, or external oblique aponeurosis. CONCLUSION: Not enough is known about the sternalis muscle to draw any conclusion about its utility in reconstructive surgery. It is hoped that cases will be presented in the foreseeable future describing its usage in reconstruction of the neck, chest, abdomen, and perhaps even other places.

Upper lip Basal cell carcinoma reconstruction based on anatomical characteristics using skin-mucosa double opposing v-y advancement flap.

BACKGROUND: Upper lip defects less than one-fourth of total upper lip length are typically closed directly, but larger defects require reconstruction. Established techniques, such as the Abbe/Estlander flap, often require multiple procedures. This report discusses a single-step method, involving a skin-mucosa double opposing V-Y advancement flap, which was utilized to reconstruct the upper lip after basal cell carcinoma extirpation. METHODS: The patient is a 54-year-old woman who presented with a 2.5-cm basal cell carcinoma in the right upper lip. Two parallel V-Y flaps were designed, on "parallel" planes of the skin and mucosa, and were advanced following wedge resection of tumor and portion of the adjacent orbicularis oris muscle. Skin/mucosa flap edges were brought to create a new skin-vermilion border, which was then sutured onto existing skin-vermilion border edges. RESULTS: The patient was evaluated 6 months after the operation, and examination showed well-healed flaps with excellent aesthetic and functional results. There was a thin, marginal scar, where reconstructed vermilion (medial) edge was sutured to native vermilion. CONCLUSIONS: It was considered that this method could reconstruct anatomical features and function of the lip precisely. It seems that within certain limits (probably between one-fourth and one-third of total upper lip length), skin-mucosa double opposing V-Y advancement flaps appear to be the preferred method for upper lip reconstruction.

Incidental positive internal mammary lymph nodes: a multiple international institutional investigation.

The internal mammary lymph node is the second most frequent site of nodal metastasis. With an increase in breast free flap reconstruction utilization of the internal mammary vessels, identification of these internal mammary lymph node metastases will become more prevalent. A chart review documented 75 free transverse rectus abdominis myocutaneous flaps cases performed at Kaiser Bellflower from 1998 to 2004. Between March 2000 and January 2006 there were 157 autologous breast reconstructions (122 DIEP flaps, 10 GAP flaps, 15 SIEA flaps, 7 free transverse rectus abdominis flaps, and 1 SCIA flap) using internal mammary vessels as the recipient site at the Chang Gung Memorial Hospital. A literature review was conducted to survey the current protocols in the surgical, oncological, and radiological communities. A population of 232 patients with breast reconstruction via free flaps was identified. The age range was 29 to 65 years. With the exception of the five cases presented, no other incidence of positive internal mammary lymph nodes was identified. Failure to account for the status of the internal mammary lymph nodes may risk understaging and preclude appropriate treatment stratification. With more frequent utilization of the internal mammary vessels, discussions regarding breast reconstruction should take this new oncological focus into consideration.

Institutional review of free TRAM flap breast reconstruction.

INTRODUCTION: A 10-year experience with breast reconstruction in a university hospital was recently reviewed. The purpose of this study was to determine the subtypes of breast reconstructive procedures and to evaluate the frequency and change in technique over time of free TRAM (transverse rectus abdominis muscle) flap breast reconstruction performed at one institution. Trends in the development of the procedure over this period were also reviewed. MATERIALS & METHODS: Between November 1994 and September 2004, a 10-year retrospective chart review was conducted. The indications for mastectomy and reconstruction were determined. The median age was 48 (range 31-66). The range of follow-up was 2 to 71 months, with a median of 19.5 months. The mean follow-up was 23.5 months. Outcome data were grouped into 2 consecutive 5-year periods (period 1: 1994-1998; period 2: 1999-2004) and evaluated for changes over time in techniques and outcome. Statistical analysis (Decision Analyst, Inc., STATS Statistics software, version 1.1, 1998) was performed using the difference between 2 proportions module to assess the probability of a significant difference in the data for period 1 and period 2 parameters. RESULTS: Over a 10-year period, 117 patients underwent breast reconstruction. This consisted of 12 pedicle procedures (11.3%), including 1 bipedicle flap (0.9%) and 2 bilateral pedicle procedures (1.8%). There were 3 latissimus dorsi pedicle flaps (2.8%). Sixteen patients (15.1%) received tissue expander or implant reconstructions. Of the 117 patients, 79 underwent free flap breast reconstruction. Of the 79 free-flap patients, 22 (27.8%) had bilateral procedures, for a total of 101 free flaps performed in these 79 patients. Fifty-two patients underwent immediate reconstruction (65.8%) and 25 were delayed (31.6%) reconstructions using either deep inferior epigastric artery perforator (DIEP) flaps (4 = 3.9%) or free TRAM flaps (97 = 96.0%). A muscle-sparing technique was used in 43 of the 97 free TRAM flaps (44.3%). The preferred vascular inflow was the internal mammary artery, which was used in 66 out of 101 flaps (65.3%). The rate of anastomotic revision (arterial and venous) was 4.9%. The majority of cases used a 2.5-mm venous coupler (65.3%). In 2 of the free TRAM cases, there was insufficient volume to establish the patients preexisting volume. Therefore, at the patient's request, immediate implants were used to augment the reconstruction. The average hospital stay was 8.13 days, and the average intensive care stay was 4.59 days. When assessed for trends over time, we noted a reduction in our hospital length of stay and our ICU length of stay. CONCLUSION: The experience with free tissue breast reconstruction reveals predominant use of the TRAM flap. This is justified by the reliability of this flap and the advances in achieving esthetic breast reconstruction. Additionally, we have begun performing DIEP free-flap reconstructions. Our clinical practice has evolved concurrent with standards of care, as noted by the increase in use of muscle-sparing techniques and the reduction in the use of dextran. We do not routinely use therapeutic anticoagulation in our cases. Our hospital length of stay and average intensive care length of stay have also decreased over time, consistent with a system-wide effort to increase the efficiency of healthcare delivery.