Effect of dulaglutide 3.0 and 4.5 mg on weight in patients with type 2 diabetes: Exploratory analyses of AWARD-11.

AIM: To evaluate the impact of dulaglutide 3.0 and 4.5 mg versus 1.5 mg on body weight in patients with type 2 diabetes (T2D) based on exploratory analyses of the AWARD-11 trial. MATERIALS AND METHODS: Patients were randomized to once-weekly dulaglutide 1.5 (n = 612), 3.0 (n = 616) or 4.5 mg (n = 614) for 52 weeks. The primary objective was superiority of dulaglutide 3.0 and/or 4.5 mg over 1.5 mg in HbA1c reduction at 36 weeks. Secondary and exploratory assessments included weight reduction in the overall trial population and baseline body mass index (BMI) and HbA1c subgroups. RESULTS: At baseline, patients had a mean age of 57.1 years, HbA1c 8.6% (70 mmol/mol), weight 95.7 kg and BMI 34.2 kg/m2 . At 36 weeks, dulaglutide 3.0 and 4.5 mg were superior to 1.5 mg for weight change from baseline (1.5 mg, -3.1 kg; 3.0 mg, -4.0 kg [P = .001]; 4.5 mg, -4.7 kg [P < .001]). Higher dulaglutide doses were associated with numerically greater weight reduction compared with 1.5 mg in each baseline BMI and HbA1c subgroup. Absolute weight reduction increased with increasing BMI category, but percentage weight loss was similar between subgroups. Weight reductions with dulaglutide were greater in patients with lower versus higher baseline HbA1c. CONCLUSIONS: In patients with T2D, inadequately controlled by metformin, incremental weight loss was observed with dulaglutide 1.5, 3.0 and 4.5 mg doses regardless of baseline BMI or HbA1c. Although absolute weight loss was numerically greater in patients with higher baseline BMI, percentage of weight loss was similar between BMI subgroups.

Bacterial collagen-like proteins that form triple-helical structures.

A large number of collagen-like proteins have been identified in bacteria during the past 10years, principally from analysis of genome databases. These bacterial collagens share the distinctive Gly-Xaa-Yaa repeating amino acid sequence of animal collagens which underlies their unique triple-helical structure. A number of the bacterial collagens have been expressed in Escherichia coli, and they all adopt a triple-helix conformation. Unlike animal collagens, these bacterial proteins do not contain the post-translationally modified amino acid, hydroxyproline, which is known to stabilize the triple-helix structure and may promote self-assembly. Despite the absence of collagen hydroxylation, the triple-helix structures of the bacterial collagens studied exhibit a high thermal stability of 35-39°C, close to that seen for mammalian collagens. These bacterial collagens are readily produced in large quantities by recombinant methods, either in the original amino acid sequence or in genetically manipulated sequences. This new family of recombinant, easy to modify collagens could provide a novel system for investigating structural and functional motifs in animal collagens and could also form the basis of new biomedical materials with designed structural properties and functions.

Defining requirements for collagenase cleavage in collagen type III using a bacterial collagen system.

Degradation of fibrillar collagens is important in many physiological and pathological events. These collagens are resistant to most proteases due to the tightly packed triple-helical structure, but are readily cleaved at a specific site by collagenases, selected members of the matrix metalloproteinases (MMPs). To investigate the structural requirements for collagenolysis, varying numbers of GXY triplets from human type III collagen around the collagenase cleavage site were inserted between two triple helix domains of the Scl2 bacterial collagen protein. The original bacterial CL domain was not cleaved by MMP-1 (collagenase 1) or MMP-13 (collagenase 3). The minimum type III sequence necessary for cleavage by the two collagenases was 5 GXY triplets, including 4 residues before and 11 residues after the cleavage site (P4-P11'). Cleavage of these chimeric substrates was not achieved by the catalytic domain of MMP-1 or MMP-13, nor by full-length MMP-3. Kinetic analysis of the chimeras indicated that the rate of cleavage by MMP-1 of the chimera containing six triplets (P7-P11') of collagen III was similar to that of native collagen III. The collagenase-susceptible chimeras were cleaved very slowly by trypsin, a property also seen for native collagen III, supporting a local structural relaxation of the triple helix near the collagenase cleavage site. The recombinant bacterial-human collagen system characterized here is a good model to investigate the specificity and mechanism of action of collagenases.

Ultrasound-guided adductor canal block for arthroscopic medial meniscectomy: a randomized, double-blind trial.

PURPOSE: The saphenous nerve block using a landmark-based approach has shown promise in reducing postoperative pain in patients undergoing arthroscopic medial meniscectomy. We hypothesized that performing an ultrasound-guided adductor canal saphenous block as part of a multimodal analgesic regimen would result in improved analgesia after arthroscopic medial meniscectomy. METHODS: Fifty patients presenting for ambulatory arthroscopic medial meniscectomy under general anesthesia were prospectively randomized to receive an ultrasound-guided adductor canal block with 0.5% ropivacaine or a sham subcutaneous injection of sterile saline. Our primary outcome was resting pain scores (numerical rating scale; NRS) upon arrival to the postanesthesia care unit (PACU). Secondary outcomes included NRS at six hours, 12 hr, 18 hr, and 24 hr; postoperative nausea; and postoperative opioid consumption. RESULTS: There was a statistically significant difference in mean NRS pain scores upon arrival to the PACU (P = 0.03): block group NRS = 1.71 (95% confidence interval [CI] 0.73 to 2.68) vs sham group NRS = 3.25 (95% CI 2.27 to 4.23). Cumulative opioid consumption (represented in oral morphine equivalents) over 24 hr was 71.8 mg (95% CI 56.5 to 87.2) in the sham group vs 44.9 mg (95% CI 29.5 to 60.2) in the block group (P = 0.016). CONCLUSIONS: An ultrasound-guided block at the adductor canal as part of a combined multimodal analgesic regimen significantly reduces resting pain scores in the PACU following arthroscopic medial meniscectomy. Furthermore, 24-hr postoperative opioid consumption and pain scores were also reduced.

Dissecting a bacterial collagen domain from Streptococcus pyogenes: sequence and length-dependent variations in triple helix stability and folding.

To better investigate the relationship between sequence, stability, and folding, the Streptococcus pyogenes collagenous domain CL (Gly-Xaa-Yaa)(79) was divided to create three recombinant triple helix subdomains A, B, and C of almost equal size with distinctive amino acid features: an A domain high in polar residues, a B domain containing the highest concentration of Pro residues, and a very highly charged C domain. Each segment was expressed as a monomer, a linear dimer, and a linear trimer fused with the trimerization domain (V domain) in Escherichia coli. All recombinant proteins studied formed stable triple helical structures, but the stability varied depending on the amino acid sequence in the A, B, and C segments and increased as the triple helix got longer. V-AAA was found to melt at a much lower temperature (31.0 °C) than V-ABC (V-CL), whereas V-BBB melted at almost the same temperature (∼36-37 °C). When heat-denatured, the V domain enhanced refolding for all of the constructs; however, the folding rate was affected by their amino acid sequences and became reduced for longer constructs. The folding rates of all the other constructs were lower than that of the natural V-ABC protein. Amino acid substitution mutations at all Pro residues in the C fragment dramatically decreased stability but increased the folding rate. These results indicate that the thermostability of the bacterial collagen is dominated by the most stable domain in the same manner as found with eukaryotic collagens.

Location of glycine mutations within a bacterial collagen protein affects degree of disruption of triple-helix folding and conformation.

The hereditary bone disorder osteogenesis imperfecta is often caused by missense mutations in type I collagen that change one Gly residue to a larger residue and that break the typical (Gly-Xaa-Yaa)(n) sequence pattern. Site-directed mutagenesis in a recombinant bacterial collagen system was used to explore the effects of the Gly mutation position and of the identity of the residue replacing Gly in a homogeneous collagen molecular population. Homotrimeric bacterial collagen proteins with a Gly-to-Arg or Gly-to-Ser replacement formed stable triple-helix molecules with a reproducible 2 °C decrease in stability. All Gly replacements led to a significant delay in triple-helix folding, but a more dramatic delay was observed when the mutation was located near the N terminus of the triple-helix domain. This highly disruptive mutation, close to the globular N-terminal trimerization domain where folding is initiated, is likely to interfere with triple-helix nucleation. A positional effect of mutations was also suggested by trypsin sensitivity for a Gly-to-Arg replacement close to the triple-helix N terminus but not for the same replacement near the center of the molecule. The significant impact of the location of a mutation on triple-helix folding and conformation could relate to the severe consequences of mutations located near the C terminus of type I and type III collagens, where trimerization occurs and triple-helix folding is initiated.

Designed coiled coils promote folding of a recombinant bacterial collagen.

Collagen triple helices fold slowly and inefficiently, often requiring adjacent globular domains to assist this process. In the Streptococcus pyogenes collagen-like protein Scl2, a V domain predicted to be largely α-helical, occurs N-terminal to the collagen triple helix (CL). Here, we replace this natural trimerization domain with a de novo designed, hyperstable, parallel, three-stranded, α-helical coiled coil (CC), either at the N terminus (CC-CL) or the C terminus (CL-CC) of the collagen domain. CD spectra of the constructs are consistent with additivity of independently and fully folded CC and CL domains, and the proteins retain their distinctive thermal stabilities, CL at ∼37 °C and CC at >90 °C. Heating the hybrid proteins to 50 °C unfolds CL, leaving CC intact, and upon cooling, the rate of CL refolding is somewhat faster for CL-CC than for CC-CL. A construct with coiled coils on both ends, CC-CL-CC, retains the ∼37 °C thermal stability for CL but shows less triple helix at low temperature and less denaturation at 50 °C. Most strikingly however, in CC-CL-CC, the CL refolds slower than in either CC-CL or CL-CC by almost two orders of magnitude. We propose that a single CC promotes folding of the CL domain via nucleation and in-register growth from one end, whereas initiation and growth from both ends in CC-CL-CC results in mismatched registers that frustrate folding. Bioinformatics analysis of natural collagens lends support to this because, where present, there is generally only one coiled-coil domain close to the triple helix, and it is nearly always N-terminal to the collagen repeat.

Efficacy and Safety of Dulaglutide 3.0 mg and 4.5 mg Versus Dulaglutide 1.5 mg in Metformin-Treated Patients With Type 2 Diabetes in a Randomized Controlled Trial (AWARD-11).

OBJECTIVE: To compare efficacy and safety of dulaglutide at doses of 3.0 and 4.5 mg versus 1.5 mg in patients with type 2 diabetes inadequately controlled with metformin. RESEARCH DESIGN AND METHODS: Patients were randomly assigned to once-weekly dulaglutide 1.5 mg, 3.0 mg, or 4.5 mg for 52 weeks. The primary objective was determining superiority of dulaglutide 3.0 mg and/or 4.5 mg over 1.5 mg in HbA1c reduction at 36 weeks. Secondary superiority objectives included change in body weight. Two estimands addressed efficacy objectives: treatment regimen (regardless of treatment discontinuation or rescue medication) and efficacy (on treatment without rescue medication) in all randomly assigned patients. RESULTS: Mean baseline HbA1c and BMI in randomly assigned patients (N = 1,842) was 8.6% (70 mmol/mol) and 34.2 kg/m2, respectively. At 36 weeks, dulaglutide 4.5 mg provided superior HbA1c reductions compared with 1.5 mg (treatment-regimen estimand: -1.77 vs. -1.54% [-19.4 vs. -16.8 mmol/mol], estimated treatment difference [ETD] -0.24% (-2.6 mmol/mol), P < 0.001; efficacy estimand: -1.87 vs. -1.53% [-20.4 vs. -16.7 mmol/mol], ETD -0.34% (-3.7 mmol/mol), P < 0.001). Dulaglutide 3.0 mg was superior to 1.5 mg for reducing HbA1c, using the efficacy estimand (ETD -0.17% [-1.9 mmol/mol]; P = 0.003) but not the treatment-regimen estimand (ETD -0.10% [-1.1 mmol/mol]; P = 0.096). Dulaglutide 4.5 mg was superior to 1.5 mg for weight loss at 36 weeks for both estimands (treatment regimen: -4.6 vs. -3.0 kg, ETD -1.6 kg, P < 0.001; efficacy: -4.7 vs. -3.1 kg, ETD -1.6 kg, P < 0.001). Common adverse events through 36 weeks included nausea (1.5 mg, 13.4%; 3 mg, 15.6%; 4.5 mg, 16.4%) and vomiting (1.5 mg, 5.6%; 3 mg, 8.3%; 4.5 mg, 9.3%). CONCLUSIONS: In patients with type 2 diabetes inadequately controlled by metformin, escalation from dulaglutide 1.5 mg to 3.0 mg or 4.5 mg provided clinically relevant, dose-related reductions in HbA1c and body weight with a similar safety profile.

Genome Characteristics Reveal the Biocontrol Potential of Actinobacteria Isolated From Sugarcane Rhizosphere.

To understand the beneficial interaction of sugarcane rhizosphere actinobacteria in promoting plant growth and managing plant diseases, this study investigated the potential role of sugarcane rhizospheric actinobacteria in promoting plant growth and antagonizing plant pathogens. We isolated 58 actinobacteria from the sugarcane rhizosphere, conducted plant growth-promoting (PGP) characteristics research, and tested the pathogenic fungi in vitro. Results showed that BTU6 (Streptomyces griseorubiginosus), the most representative strain, regulates plant defense enzyme activity and significantly enhances sugarcane smut resistance by regulating stress resistance-related enzyme (substances (POD, PAL, PPO, TP) in sugarcane) activity in sugarcane. The genomic evaluation indicated that BTU6 has the ability to biosynthesize chitinase, ß-1,3-glucanase, and various secondary metabolites and plays an essential role in the growth of sugarcane plants under biotic stress. Potential mechanisms of the strain in improving the disease resistance of sugarcane plants and its potential in biodegrading exogenous chemicals were also revealed. This study showed the importance of sugarcane rhizosphere actinobacteria in microbial ecology and plant growth promotion.

Expanding the family of collagen proteins: recombinant bacterial collagens of varying composition form triple-helices of similar stability.

The presence of the (Gly-Xaa-Yaa)(n) open reading frames in different bacteria predicts the existence of an expanded family of collagen-like proteins. To further explore the triple-helix motif and stabilization mechanisms in the absence of hydroxyproline (Hyp), predicted novel collagen-like proteins from Gram-positive and -negative bacteria were expressed in Escherichia coli and characterized. Soluble proteins capable of successful folding and in vitro refolding were observed for collagen proteins from Methylobacterium sp 4-46, Rhodopseudomonas palustris and Solibacter usitatus . In contrast, all protein constructs from Clostridium perfringens were found predominantly in inclusion bodies. However, attachment of a heterologous N-terminal or C-terminal noncollagenous folding domain induced the Clostridium perfringens collagen domain to fold and become soluble. The soluble constructs from different bacteria had typical collagen triple-helical features and showed surprisingly similar thermal stabilities despite diverse amino acid compositions. These collagen-like proteins provide a resource for the development of biomaterials with new properties.

Draft Genome Analysis Offers Insights Into the Mechanism by Which Streptomyces chartreusis WZS021 Increases Drought Tolerance in Sugarcane.

Drought directly affects sugarcane production. Plant growth-promoting bacteria have gained attention as growth promoters of plants under abiotic stresses. The present study focused on genome assessment of the plant-beneficial endophyte Streptomyces chartreusis WZS021 and its vital role in sugarcane plants under drought stress. Based on in vitro plant growth-promoting trait analyses, WZS021 had multiple abilities, including tolerance to drought and production of 1-aminocyclopropane-1-carboxylic deaminase, siderophores, and indole acetic acid. We confirmed root colonization of sugarcane transplants by WZS021 by a sterile sand assay and scanning electron microscopy. Plants inoculated with strain WZS021 had a positive influence on the root parameters such as length and biomass when compared to the control plants. A comparative study of the responses of two sugarcane varieties (ROC22 and B8) to different levels of drought stress in the presence or absence of WZS021 was conducted by assessing the plant chemistry. The expression of antioxidants in sugarcane leaves varied with water stress level. WZS021 inoculation improved the contents of chlorophyll, proline, and phytohormones, revealing some potential for the mechanisms by which this strain improves drought tolerance in sugarcane plants. We identified several genes that might be involved in the plant growth- and drought tolerance-promoting effects of this strain.

N-of-1 Design and Its Applications to Personalized Treatment Studies.

N-of-1 trial is a type of clinical trial which has been applied in chronic recurrent conditions that require long-term non-curative treatment. In this type of trials, each patient will be randomly assigned to one of the treatment sequences and repeatedly crossed over two or more treatments of interests. Through this cross-comparing method (cross-over phase), investigator can identify an optimal treatment (medicine or therapy) for the patient and treat the patient with the optimal treatment in an extension phase. This design could efficiently reduce the placebo effect, which is often seen in clinical trials, and maximize the true treatment effect. This type of design has been used in some traditional Chinese medicine (TCM) clinical trials lately. However, it brings some challenges for collecting and analyzing the data. Research on statistical methodology of this type of design is rarely found in the literature. The goal of this research is to discuss the application of the N-of-1 design to personalized treatment studies. We will demonstrate a real study conducted in TCM and present some theoretical and simulation results.

Noncollagenous region of the streptococcal collagen-like protein is a trimerization domain that supports refolding of adjacent homologous and heterologous collagenous domains.

Proper folding of the (Gly-Xaa-Yaa)(n) sequence of animal collagens requires adjacent N- or C-terminal noncollagenous trimerization domains which often contain coiled-coil or beta sheet structure. Collagen-like proteins have been found recently in a number of bacteria, but little is known about their folding mechanism. The Scl2 collagen-like protein from Streptococcus pyogenes has an N-terminal globular domain, designated V(sp), adjacent to its triple-helix domain. The V(sp) domain is required for proper refolding of the Scl2 protein in vitro. Here, recombinant V(sp) domain alone is shown to form trimers with a significant alpha-helix content and to have a thermal stability of T(m) = 45 degrees C. Examination of a new construct shows that the V(sp) domain facilitates efficient in vitro refolding only when it is located N-terminal to the triple-helix domain but not when C-terminal to the triple-helix domain. Fusion of the V(sp) domain N-terminal to a heterologous (Gly-Xaa-Yaa)(n) sequence from Clostridium perfringens led to correct folding and refolding of this triple-helix, which was unable to fold into a triple-helical, soluble protein on its own. These results suggest that placement of a functional trimerization module adjacent to a heterologous Gly-Xaa-Yaa repeating sequence can lead to proper folding in some cases but also shows specificity in the relative location of the trimerization and triple-helix domains. This information about their modular nature can be used in the production of novel types of bacterial collagen for biomaterial applications.

A Streptococcus pyogenes derived collagen-like protein as a non-cytotoxic and non-immunogenic cross-linkable biomaterial.

A range of bacteria have been shown to contain collagen-like sequences that form triple-helical structures. Some of these proteins have been shown to form triple-helical motifs that are stable around body temperature without the inclusion of hydroxyproline or other secondary modifications to the protein sequence. This makes these collagen-like proteins particularly suitable for recombinant production as only a single gene product and no additional enzyme needs to be expressed. In the present study, we have examined the cytotoxicity and immunogenicity of the collagen-like domain from Streptococcus pyogenes Scl2 protein. These data show that the purified, recombinant collagen-like protein is not cytotoxic to fibroblasts and does not elicit an immune response in SJL/J and Arc mice. The freeze dried protein can be stabilised by glutaraldehyde cross-linking giving a material that is stable at >37 degrees C and which supports cell attachment while not causing loss of viability. These data suggest that bacterial collagen-like proteins, which can be modified to include specific functional domains, could be a useful material for medical applications and as a scaffold for tissue engineering.

Self-association of streptococcus pyogenes collagen-like constructs into higher order structures.

A number of bacterial collagen-like proteins with Gly as every third residue and a high Pro content have been observed to form stable triple-helical structures despite the absence of hydroxyproline (Hyp). Here, the high yield cold-shock expression system is used to obtain purified recombinant collagen-like protein (V-CL) from Streptococcus pyogenes containing an N-terminal globular domain V followed by the collagen triple-helix domain CL and the modified construct with two tandem collagen domains V-CL-CL. Both constructs and their isolated collagenous domains form stable triple-helices characterized by very sharp thermal transitions at 35-37 degrees C and by high values of calorimetric enthalpy. Procedures for the formation of collagen SLS crystallites lead to parallel arrays of in register V-CL-CL molecules, as well as centrosymmetric arrays of dimers joined at their globular domains. At neutral pH and high concentrations, the bacterial constructs all show a tendency towards aggregation. The isolated collagen domains, CL and CL-CL, form units of diameter 4-5 nm which bundle together and twist to make larger fibrillar structures. Thus, although this S. pyogenes collagen-like protein is a cell surface protein with no indication of participation in higher order structure, the triple-helix domain has the potential of forming fibrillar structures even in the absence of hydroxyproline. The formation of fibrils suggests bacterial collagen proteins may be useful for biomaterials and tissue engineering applications.