Search

Dentistry has evolved from its origin to the present day, becoming almost entirely digitized and supervised. The digitalized dental laboratory saves time due to computer-aided design and computer-aided manufacture (CAD/CAM) technology, which will capture and display clients' tooth or teeth and gums on a 3D image on a computer screen sent to the lab. It enables a dental lab technician to work faster and get the perfect design of the digital dental restoration. The main advantage of digitalization includes faster and improved efficiency on the turn-around time of devices, like crowns and bridges, and improved accuracy of procedures and manufactured gadgets. Digitalization Dental Laboratory (DDL) is the first to offer a digital dental lab in the city of Bacolod. The service allows laboratories to design the prosthesis digitally from in-house CAD software and email the design data provider or download the data file into a proprietary web host or server. The lab will cater to the digital needs of dental patients of the Multi-Specialty Dental Center (a sister company of DDL) and other dental clients.

Both central and peripheral cannabinoid receptor type 1 (CB1R) have been considered to be among the key targets for obesity treatment. First generation CB1R antagonists/inverse agonists such as rimonabant and taranabant exhibited severe CNS side effects such as anxiety and depression, which are considered to be related to the compounds' ability to access central CB1R. Recently, several compounds have been developed as second generation antagonists with a profile of restriction to peripheral CB1R. We evaluated the distribution of TM38837, a second generation CB1R antagonist, using brain and whole body PET in three cynomolgus monkeys, and established the relationship between CB1R occupancy and dose/plasma concentration of TM38837 in comparison with rimonabant. A brain PET study was performed using [(11) C]MePPEP, a PET radioligand for CB1R, to evaluate the brain CB1R occupancy of TM38837 at various plasma concentrations in comparison with rimonabant at known efficacious plasma concentrations. A whole body PET study was performed to investigate the change of peripheral distribution of [(11) C]MePPEP by TM38837 administration, which indirectly estimated the effects to the peripheral CB1R by TM38837. CB1R occupancy by both TM38837 and rimonabant increased in a dose/plasma concentration-dependent manner. However, in vivo affinity by plasma level was more than 100 times lower for TM38837. Peripherally, [(11) C]MePPEP accumulation decreased in gall bladder and brown adipose tissue by TM38837 administration. TM38837 showed rather lower CB1R occupancy than rimonabant at the expected therapeutic plasma level, which is expected to reduce CNS side effects in clinical situations. Further clinical development of TM38837 is warranted.

Aim Cannabinoid receptor type 1 (CB1) antagonists show central side effects, whereas beneficial effects are most likely peripherally mediated. In this study, the peripherally selective CB1 antagonist TM38837 was studied in humans. Methods This was a double-blind, randomized, placebo-controlled, crossover study. On occasions 1–4, 24 healthy subjects received 5 × 4 mg THC with TM38837 100 mg, 500 mg or placebo, or placebos only. During occasion 5, subjects received placebo TM38837 + THC with rimonabant 60 mg or placebo in parallel groups. Blood collections and pharmacodynamic (PD) effects were assessed frequently. Pharmacokinetics (PK) and PD were quantified using population PK−PD modelling. Results The TM38837 plasma concentration profile was relatively flat compared with rimonabant. TM38837 showed an estimated terminal half-life of 771 h. THC induced effects on VAS feeling high, body sway and heart rate were partly antagonized by rimonabant 60 mg [−26.70% [90% confidence interval (CI) −40.9, −12.6%]; −7.10%, (90%CI −18.1, 5.3%); −7.30%, (90% CI −11.5%, −3.0%) respectively] and TM38837 500 mg [−22.10% (90% CI −34.9, −9.4%); −12.20% (90% CI −21.6%, −1.7%); −8.90% (90% CI −12.8%, −5.1%) respectively]. TM38837 100 mg had no measurable feeling high or body sway effects and limited heart rate effects. Conclusions Rimonabant showed larger effects than TM38837, but the heart rate effects were similar. TM38837 100 mg had no impact on CNS effects, suggesting that this dose does not penetrate the brain. This TM38837 dose is predicted to be at least equipotent to rimonabant with regard to metabolic disorders in rodent models. These results provide support for further development of TM38837 as a peripherally selective CB1 antagonist for indications such as metabolic disorders, with a reduced propensity for psychiatric side effects.

A 28-year-old man presented emergently to the operating room following a gun-shot injury to his right groin. Our hospital’s Massive Transfusion Protocol was initiated as the patient entered the operating room actively hemorrhaging and severely hypotensive. During the aggressive resuscitation efforts, the patient was inadvertently transfused 2 units of packed red blood cells intended for another patient due to a series of errors. Fortunately, the incorrect product was compatible, and the patient recovered from his near-fatal injuries. Root cause analysis was used to review the transfusion error and develop an action plan to help prevent future occurrences.

Genetic engineering has been applied to reprogramme non-ribosomal peptide synthetases (NRPSs) to produce novel antibiotics, but little is known about what determines the efficiency of production. We explored module exchanges at nucleotide sequences encoding interpeptide linkers in dptD, a gene encoding a di-modular NRPS subunit that incorporates 3-methylglutamic acid (3mGlu(12)) and kynurenine (Kyn(13)) into daptomycin. Mutations causing amino acid substitutions, deletions or insertions in the inter-module linker had no negative effects on lipopeptide yields. Hybrid DptD subunits were generated by fusing the 3mGlu(12) module to terminal modules from calcium-dependent antibiotic (CDA) or A54145 NRPSs, and recombinants produced daptomycin analogues with Trp(13) or Ile(13) at high efficiencies. A recombinant expressing DptD with a hybrid Kyn(13) module containing a di-domain from a d-Asn module caused the production of a new daptomycin analogue containing Asn(13).

Daptomycin, a cyclic lipopeptide produced by Streptomyces roseosporus , is the active ingredient of Cubicin (daptomycin-for-injection), a first-in-class antibiotic approved for treatment of skin and skin-structure infections caused by Gram-positive pathogens and bacteremia and endocarditis caused by Staphylococcus aureus , including methicillin-resistant strains. Genetic engineering of the nonribosomal peptide synthetase (NRPS) in the daptomycin biosynthetic pathway was exploited for the biosynthesis of novel active antibiotics. λ-Red-mediated recombination was used to exchange single or multiple modules in the DptBC subunit of the NRPS to modify the daptomycin cyclic peptide core. We combined module exchanges, NRPS subunit exchanges, inactivation of the tailoring enzyme glutamic acid 3-methyltransferase, and natural variations of the lipid tail to generate a library of novel lipopeptides, some of which were as active as daptomycin against Gram-positive bacteria. One compound was more potent against an Escherichia coli imp mutant that has increased outer membrane permeability. This study established a robust combinatorial biosynthesis platform to produce novel peptide antibiotics in sufficient quantities for antimicrobial screening and drug development.

SummaryDaptomycin is a lipopeptide antibiotic produced by a nonribosomal peptide synthetase (NRPS) in Streptomyces roseosporus. The holoenzyme is composed of three subunits, encoded by the dptA, dptBC, and dptD genes, each responsible for incorporating particular amino acids into the peptide. We introduced expression plasmids carrying dptD or NRPS genes encoding subunits from two related lipopeptide biosynthetic pathways into a daptomycin nonproducing strain of S. roseosporus harboring a deletion of dptD. All constructs successfully complemented the deletion in trans, generating three peptide cores related to daptomycin. When these were coupled with incomplete methylation of 1 amino acid and natural variation in the lipid side chain, 18 lipopeptides were generated. Substantial amounts of nine of these compounds were readily obtained by fermentation, and all displayed antibacterial activity against gram-positive pathogens.

Daptomycin and the A21978C antibiotic complex are lipopeptides produced by Streptomyces roseosporus and also in recombinant Streptomyces lividans TK23 and TK64 strains, when a 128 kbp region of cloned S. roseosporus DNA containing the daptomycin gene cluster is inserted site-specifically in the phiC31 attB site. A21978C fermentation yields were initially much lower in S. lividans than in S. roseosporus, and detection was complicated by the production of host metabolites. However A21978C production in S. lividans was improved by deletion of genes encoding the production of actinorhodin and by medium optimization to control the chemical form of the calcium dependent antibiotic (CDA). This latter compound has not previously been chemically characterized as a S. lividans product. Adding phosphate to a defined fermentation medium resulted in formation of only the phosphorylated forms of CDA, which were well separated from A21978C on chromatographic analysis. Adjusting the level of phosphate in the medium led to an improvement in A21978C yield from 20 to 55 mg/l.

Daptomycin is a cyclic lipopeptide antibiotic produced by Streptomyces roseosporus. Cubicin (daptomycin-for-injection) was approved in 2003 by the FDA to treat skin and skin structure infections caused by Gram-positive pathogens. Daptomycin is particularly significant in that it represents the first new natural product antibacterial structural class approved for clinical use in three decades. The daptomycin gene cluster contains three very large genes (dptA, dptBC, and dptD) that encode the nonribosomal peptide synthetase (NRPS). The related cyclic lipopeptide A54145 has four NRPS genes (lptA, lptB, lptC, and lptD), and calcium dependent antibiotic (CDA) has three (cdaPS1, cdaPS2, and cdaPS3). Mutants of S. roseosporus containing deletions of one or more of the NRPS genes have been trans-complemented with dptA, dptBC, and dptD by inserting these genes under the control of the ermEp* promoter into separate conjugal cloning vectors containing phiC31 or IS117 attachment (attP int) sites; delivering the plasmids into S. roseosporus by conjugation from Escherichia coli; and inserting the plasmids site-specifically into the chromosome at the corresponding attB sites. This trans-complementation system was used to generate subunit exchanges with lptD and cdaPS3 and the recombinants produced novel hybrid molecules. Module exchanges at positions D: -Ala(8) and D: -Ser(11) in the peptide have produced additional novel derivatives of daptomycin. The approaches of subunit exchanges and module exchanges were combined with amino acid modifications of Glu at position 12 and natural variations in lipid side chain starter units to generate a combinatorial library of antibiotics related to daptomycin. Many of the engineered strains produced levels of novel molecules amenable to isolation and antimicrobial testing, and most of the compounds displayed antibacterial activities.

Daptomycin is a 13 amino acid, cyclic lipopeptide produced by a non-ribosomal peptide synthetase (NRPS) mechanism inStreptomyces roseosporus. A 128 kb region ofS. roseosporusDNA was cloned and verified by heterologous expression inStreptomyces lividansto contain the daptomycin biosynthetic gene cluster (dpt). The cloned region was completely sequenced and three genes (dptA,dptBC,dptD) encoding the three subunits of an NRPS were identified. The catalytic domains in the subunits, predicted to couple five, six or two amino acids, respectively, included a novel activation domain and amino-acid-binding pocket for incorporating the unusual amino acidl-kynurenine (Kyn), three types of condensation domains and an extra epimerase domain (E-domain) in the second module. Novel genes (dptE,dptF) whose products likely work in conjunction with a unique condensation domain to acylate the first amino acid, as well as other genes (dptI,dptJ) probably involved in supply of the non-proteinogenic amino acidsl-3-methylglutamic acid and Kyn, were located next to the NRPS genes. The unexpected E-domain suggested that daptomycin would haved-Asn, rather thanl-Asn, as originally assigned, and this was confirmed by comparing stereospecific synthetic peptides and the natural product both chemically and microbiologically.

Takayasu’s arteritis is a rare, progressive panendarteritis involving all layers of the arterial wall. This disease includes variable involvement of the aorta and its major branches. The most common complication with this condition is severe, uncontrolled hypertension, often leading to end organ dysfunction. We describe the management of a 27-year-old woman diagnosed with Takayasu’s arteritis that presented in labor with intense pain and underwent a combined spinal-epidural for anesthetic management. Per literature review, a combined spinal-epidural technique for planned vaginal delivery has not been described for a laboring Takayasu patient. Our technique, utilizing intrathecal opioids and a low-dose local anesthetic-opioid epidural infusion, provided adequate analgesia while maintaining hemodynamic stability throughout labor augmentation and successful vaginal delivery.

A potent new lipopeptide antibiotic, A54145E(Asn(3)Asp(9)), was isolated from the fermentation broth of Streptomyces fradiae DA1489 engineered to delete genes encoding enzymes involved in hydroxylation of Asn(3) and methoxylation of Asp(9). The chemical structure predicted from the genetic changes in the biosynthetic pathway was determined by analyses of chemical transformations, D, L-amino acid quantitation by enantiomer labeling, tandem LC-MS/MS and 2D NMR techniques. These studies confirmed the primary amino acid sequence of A54145E(Asn(3)Asp(9)) predicted from the genetic engineering strategy, and also confirmed the structure and locations of three D-amino acids predicted from bioinformatic studies.

A54145 is a complex of lipopeptide antibiotics produced by Streptomyces fradiae. A54145 factors are structurally related to daptomycin, with four modified amino acids, only one of which is present in daptomycin. We generated three mutants defective in lptJ, lptK or lptL, whose gene products are involved in the formation of hydroxy-Asn(3) (hAsn(3)) and methoxy-Asp(9) (moAsp(9)). Each of the mutants produced novel lipopeptides related to A54145 and the profiles allowed assignment of functions for those genes. We constructed strains carrying different combinations of these genes coupled with a mutation in the lptI gene involved in the biosynthesis of 3-methyl-Glu(12) (3mGlu(12)), and all recombinants produced novel lipopeptides. One of the compounds displayed very good antibacterial activity in the presence of bovine surfactant, which interacts with daptomycin or A54145E to inhibit their antibacterial activities.

Three daptomycin-related lipopeptides, A21978C1-3(d-Asn11) (2-4), were purified from the fermentation broth of a recombinant Streptomyces roseosporus strain. Their chemical structures were determined by analyses of the biosynthetic pathway, chemical transformations, d,l-amino acid quantitation by enantiomer labeling, tandem LC-MS/MS, and 2D-NMR techniques. Compounds 2-4 exhibited potent antibacterial activity against Staphylococcus aureus with MIC values of 0.6, 0.3, and 0.15 microM, respectively, well correlated to the acyl tail chain length.

Aluminum toxicity in acid soils severely limits crop productivity through inhibition of root growth and, consequently, shoot development. Several Arabidopsis mutants were previously identified as having roots with Al hypersensitivity, suggesting that these represent deleterious mutations affecting genes required for either Al tolerance or resistance mechanisms. For this report, the als1-1 mutant was chosen for further characterization. The phenotype of als1-1 is most obviously presented in Al challenged roots, as evidenced by exaggerated root growth inhibition in conjunction with increased expression of Al-responsive genes compared to wt. Using a map-based cloning approach, the als1-1 mutation was isolated and found to represent a deleterious amino acid substitution in a previously uncharacterized half type ABC transporter, At5g39040, which is expressed in a non-Al dependent manner in all organs tested. GUS-dependent analyses revealed that ALS1 expression is primarily localized to the root tip and the vasculature throughout the plant. Concomitant with this, an ALS1: GFP fusion accumulates at the vacuolar membrane of root cells, indicating that ALS1 may be important for intracellular movement of some substrate, possibly chelated Al, as part of a mechanism of Al sequestration.

In many peptide antibiotics, modified amino acids are important for biological activity. The amino acid 3-methyl-glutamic acid (3mGlu) has been found only in three cyclic lipopeptide antibiotics: daptomycin and the A21978C family produced by Streptomyces roseosporus, calcium-dependent antibiotic produced by Streptomyces coelicolor and A54145 produced by Streptomyces fradiae. We studied the non-ribosomal peptide synthetase genes involved in A21978C biosynthesis and the downstream genes, dptG, dptH, dptI and dptJ predicted to encode a conserved protein of unknown function, a thioesterase, a methyltransferase (MTase) and a tryptophan 2,3-dioxygenase respectively. Deletion of dptGHIJ reduced overall lipopeptide yield and led to production of a series of novel A21978C analogues containing Glu12 instead of 3mGlu12. Complementation by only dptI, or its S. coelicolor homologue, glmT, restored the biosynthesis of the 3mGlu-containing compounds in the mutant. Compared with A21978C, the Glu12-containing derivatives were less active against Staphylococcus aureus. Further genetic analyses showed that members of the dptGHIJ locus cooperatively contributed to optimal A21978C production; deletion of dptH, dptI or dptJ genes reduced the yield significantly, while expression of dptIJ or dptGHIJ from the strong ermEp* promoter substantially increased lipopeptide production. The results indicate that these genes play important roles in the biosynthesis of daptomycin, and that dptI encodes a Glu MTase.