Impacts of Hydrophobic Mismatch on Antimicrobial Peptide Efficacy and Bilayer Permeabilization.

Antimicrobial resistance continues to be a major threat to world health, with the continued emergence of resistant bacterial strains. Antimicrobial peptides have emerged as an attractive option for the development of novel antimicrobial compounds in part due to their ubiquity in nature and the general lack of resistance development to this class of molecules. In this work, we analyzed the antimicrobial peptide C18G and several truncated forms for efficacy and the underlying mechanistic effects of the sequence truncation. The peptides were screened for antimicrobial efficacy against several standard laboratory strains, and further analyzed using fluorescence spectroscopy to evaluate binding to model lipid membranes and bilayer disruption. The results show a clear correlation between the length of the peptide and the antimicrobial efficacy. Furthermore, there is a correlation between peptide length and the hydrophobic thickness of the bilayer, indicating that hydrophobic mismatch is likely a contributing factor to the loss of efficacy in shorter peptides.

Effect of cell culture medium additives on color and acidic charge variants of a monoclonal antibody.

This manuscript summarizes the effect of certain cell culture medium additives on antibody drug substance coloration and acidic charge variants. It has been shown previously that B-vitamins and iron in the cell culture medium could significantly impact color intensity. In this manuscript, we detail the effect of several other cell culture components that have been shown to impact coloration. It is shown that if cystine is used instead of cysteine in the cell culture medium, coloration was reduced. Hydrocortisone has been shown to reduce coloration and boost specific productivity. The effect of a peptone/hydrolysate on coloration was investigated in cell culture experiments, which showed its use can lead to reduced coloration. Mechanisms by which these compounds influence coloration will be briefly discussed. Since it has been previously shown that antibody oxidation could potentially lead to coloration, the current effort was focused on screening for specific antioxidant additives to the culture medium to reduce coloration. An in-vitro incubation model was used to screen antioxidant compounds, several of which were found to significantly reduce antibody color, while some led to significantly increased color. Hypotaurine and carboxymethylcysteine, which had the most significant color reducing effect in the incubation study, were further tested in small-scale bioreactor cell culture experiments. These studies demonstrated that these compounds lead to reduced coloration in cell culture without affecting cell growth and titer. Hypotaurine, hydrocortisone, peptone, and cystine were also shown to reduce the acidic charge variant levels, which was previously shown to correlate with color. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018 © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1298-1307, 2018.

Process characterization and Design Space definition.

Quality by design (QbD) is a global regulatory initiative with the goal of enhancing pharmaceutical development through the proactive design of pharmaceutical manufacturing process and controls to consistently deliver the intended performance of the product. The principles of pharmaceutical development relevant to QbD are described in the ICH guidance documents (ICHQ8-11). An integrated set of risk assessments and their related elements developed at Roche/Genentech were designed to provide an overview of product and process knowledge for the production of a recombinant monoclonal antibody (MAb). This chapter describes the tools used for the characterization and validation of MAb manufacturing process under the QbD paradigm. This comprises risk assessments for the identification of potential Critical Process Parameters (pCPPs), statistically designed experimental studies as well as studies assessing the linkage of the unit operations. Outcome of the studies is the classification of process parameters according to their criticality and the definition of appropriate acceptable ranges of operation. The process and product knowledge gained in these studies can lead to the approval of a Design Space. Additionally, the information gained in these studies are used to define the 'impact' which the manufacturing process can have on the variability of the CQAs, which is used to define the testing and monitoring strategy.

Exploring the potential of fulvalene dimetals as platforms for molecular solar thermal energy storage: computations, syntheses, structures, kinetics, and catalysis.

A study of the scope and limitations of varying the ligand framework around the dinuclear core of FvRu2 in its function as a molecular solar thermal energy storage framework is presented. It includes DFT calculations probing the effect of substituents, other metals, and CO exchange for other ligands on ΔHstorage . Experimentally, the system is shown to be robust in as much as it tolerates a number of variations, except for the identity of the metal and certain substitution patterns. Failures include 1,1',3,3'-tetra-tert-butyl (4), 1,2,2',3'-tetraphenyl (9), diiron (28), diosmium (24), mixed iron-ruthenium (27), dimolybdenum (29), and ditungsten (30) derivatives. An extensive screen of potential catalysts for the thermal reversal identified AgNO3 -SiO2 as a good candidate, although catalyst decomposition remains a challenge.

Implementing high-temperature short-time media treatment in commercial-scale cell culture manufacturing processes.

The production of therapeutic proteins by mammalian cell culture is complex and sets high requirements for process, facility, and equipment design, as well as rigorous regulatory and quality standards. One particular point of concern and significant risk to supply chain is the susceptibility to contamination such as bacteria, fungi, mycoplasma, and viruses. Several technologies have been developed to create barriers for these agents to enter the process, e.g. filtration, UV inactivation, and temperature inactivation. However, if not implemented during development of the manufacturing process, these types of process changes can have significant impact on process performance if not managed appropriately. This article describes the implementation of the high-temperature short-time (HTST) treatment of cell culture media as an additional safety barrier against adventitious agents during the transfer of a large-scale commercial cell culture manufacturing process. The necessary steps and experiments, as well as subsequent results during qualification runs and routine manufacturing, are shown.

Single-molecule-resolved structural changes induced by temperature and light in surface-bound organometallic molecules designed for energy storage.

We have used scanning tunneling microscopy, Auger electron spectroscopy, and density functional theory calculations to investigate thermal and photoinduced structural transitions in (fulvalene)tetracarbonyldiruthenium molecules (designed for light energy storage) on a Au(111) surface. We find that both the parent complex and the photoisomer exhibit striking thermally induced structural phase changes on Au(111), which we attribute to the loss of carbonyl ligands from the organometallic molecules. Density functional theory calculations support this conclusion. We observe that UV exposure leads to pronounced structural change only in the parent complex, indicative of a photoisomerization reaction.

Experimental realization of catalytic CH4 hydroxylation predicted for an iridium NNC pincer complex, demonstrating thermal, protic, and oxidant stability.

A discrete, air, protic, and thermally stable (NNC)Ir(III) pincer complex was synthesized that catalytically activates the CH bond of methane in trifluoroacetic acid; functionalization using NaIO4 and KIO3 gives the oxy-ester.

Over-pointing technique: an approach to past-pointing arthroscopic knots on alternating suture posts without alternating the knot pusher.

As arthroscopic reconstructive surgery evolves, new techniques that improve operative efficiency while maintaining proven technical principles will enhance a surgeon's skill repertoire. A secure arthroscopic knot requires placing a series of reversed half-hitches on alternating posts and has traditionally involved alternating the knot-passing device between suture limbs to "past-point" each half-hitch, ensuring overall knot security. To increase the speed of knot-tying, a technical variation of keeping the knot pusher on the same suture limb throughout the knot-tying process, while still alternating posts, can be used. This technique involves alternately "pushing" and "pulling" sequential half-hitches into place. It is important to note that although conventional past-pointing is performed when "pulling" a half-hitch, it cannot be performed when purely "pushing" a half-hitch. In this article we describe a novel technique of "over-pointing" to be performed when pushing half-hitches into place. This is meant to be a complement to past-pointing by use of the knot pusher to advance the half-hitch and tighten the knot by over-riding the knot, effectively providing the equivalent of past-pointing by "pulling" the knot tight. In this way, a knot can be constructed without removing the device from the initial post strand of suture, saving time while maintaining the principles of secure knot-tying.

The effect of double-row fixation on initial repair strength in rotator cuff repair: a biomechanical study.

PURPOSE: The purpose of this study was to compare the initial mechanical strength of 3 rotator cuff repair techniques. METHODS: A total of 30 fresh-frozen cadaveric shoulders were prepared, and full-thickness supraspinatus tears were created. Specimens were randomized and placed into 3 groups: (1) transosseous suture technique (group I: TOS, n = 10, 6F/4M), (2) single-row suture anchor fixation (group II: SRSA, n = 10, 6F/4M), and (3) double-row suture anchor fixation (group III: DRSA, n = 10, 6F/4M). Each specimen underwent cyclic load testing from 5 N to 180 N at a rate of 33 mm/sec. The test was stopped when complete failure (repair site gap of 10 mm) or a total of 5,000 cycles was attained. RESULTS: Group I (TOS) failed at an average of 75.3 +/- 22.49 cycles, and group II (SRSA) at an average of 798.3 +/- 73.28 cycles; group III (DRSA) had no failures because all samples were stopped when 5,000 cycles had been completed. Fixation strength of the DRSA technique proved to be significantly greater than that of SRSA (P < .001), and both suture anchor groups were significantly stronger than the TOS group (P < .001). CONCLUSIONS: Suture anchor repairs were significantly stronger than transosseous repairs. Furthermore, double-row suture anchor fixation was significantly stronger than was single-row repair. Therefore, double-row fixation may be superior to other techniques in that it provides a substantially stronger repair that could lead to improved biologic healing. CLINICAL RELEVANCE: A high incidence of incomplete healing occurs in rotator cuff repair. Use of double-row fixation may help the clinician to address some deficiencies in current methods by increasing the strength of the repair, potentially leading to improved healing rates.

Rotator cuff repair: the effect of double-row fixation on three-dimensional repair site.

There is a high rate of recurrent and residual tears after rotator cuff repair surgery. Recent cadaveric studies have provided surgeons with new knowledge about the anatomy of the supraspinatus tendon insertion. Traditional repair techniques fail to reproduce the area of the supraspinatus insertion, or footprint, on the greater tuberosity anatomically. Double-row suture anchor (DRSA) fixation is a new technique that has been developed to restore the supraspinatus footprint better. In this study, 3-dimensional mapping was used to determine the area of the footprint recreated with 3 different repair methods: a transosseous simple suture technique, fixation with a single row of suture anchors, and DRSA fixation. The DRSA fixation technique consistently reproduced 100% of the original supraspinatus footprint, whereas the single-row suture anchor fixation and transosseous simple suture techniques reproduced only 46% and 71% of the insertion site, respectively. Therefore, the footprint area of the DRSA fixation technique was significantly larger (P < .05) than that of the other 2 techniques. Furthermore, double-row fixation may provide a tendon-bone interface better suited for biologic healing and restoring normal anatomy.

Capturing patterns and symmetries in chaotic granular flow.

Segregation patterns formed by time-periodic flow of polydisperse granular material (varying in particle size) in quasi-two-dimensional (quasi-2D) tumblers capture the symmetries of Poincaré sections, stroboscopic maps of the underlying flow, derived from a continuum model which contains no information about particle properties. We study this phenomenon experimentally by varying the concentration of small particles in a bidisperse mixture in quasi-2D tumblers with square and pentagonal cross sections. By coupling experiments with an analysis of periodic points, we explain the connection between the segregation patterns and the dynamics of the underlying flow. Analysis of the eigenvectors and unstable manifolds of hyperbolic points shows that lobes of segregated small particles stretch from hyperbolic points toward corners of the tumbler, demonstrating the connection between regions of chaotic flow and the shape of the segregation patterns. Furthermore, unstable manifolds map the shape of lobes of segregated particles. The techniques developed here can also be applied to nonpolygonal tumblers such as elliptical tumblers, as well as to circular tumblers with time-periodic forcing.

Radiation characteristics of a high-emissivity cylindrical-spherical cavity with obscuration.

We have calculated, to first order, the apparent emissivity of the bounding diffuse surfaces of a high-emissivity cylindrical-spherical cavity enclosure. Our calculations indicate that to achieve emissivities close to a perfectly absorbing blackbody cavity along the bounding surfaces of the spherical enclosure, the radius of the sphere must be equal to or greater than a factor of 4 times the cylinder radius R(S) > or = 4R(C). Furthermore, to achieve emissivities approaching a blackbody cavity along the lower bounding surfaces of the cylindrical enclosure, the length of the cylinder must be a factor of 4 times greater than the radius of the cylinder L > or = 4R(C). In addition, we present the mathematical framework necessary to calculate radiant transfer within a cavity enclosure that contains obscuration. These results can be applied to the design of high-emissivity blackbody calibration cavities and to the reduction of stray light in terrestrial and spaceborne optical systems.

Approach to cartilage injury in the anterior cruciate ligament-deficient knee.

The treatment of articular cartilage lesions remains one of the great challenges facing orthopedic surgeons today. The technique of chondrocyte transplantation has opened the door for the application of biologic solutions to difficult problems. These techniques will prove the keystone of further advances into biologic joint repair and replacement. Enthusiasm, however, must be tempered by the numerous gaps in cartilage science and the overwhelming need for further long-term data to demonstrate the efficacy of these techniques in thwarting the presumed eventual progression of these lesions toward osteoarthritis. The status of the articular cartilage is of paramount importance in ACL decision-making. Every effort must be made to protect the existing hyaline articular cartilage during ACL reconstruction. Though current cartilage repair techniques are in their infancy, they remain stepping-stones to future developments. It is hoped that we will one day be able to regenerate normal hyaline cartilage without great morbidity. At present, the ACL surgeon must accept techniques that diminish symptoms and do not burn bridges to future advances. The orthopedic surgeon must increase his knowledge of the basic science of articular cartilage in order to best choose from the various cartilage treatments that evolve.