Protein Particulate Detection Issues in Biotherapeutics Development—Current Status

Formation of aggregates and particulates in biopharmaceutical formulation continues to be one of the major quality concerns in biotherapeutics development. The presence of large quantities of aggregates is believed to be one of the causes of unwanted immunogenic responses. Protein particulates can form in a wide range of sizes and shapes. Therefore, a comprehensive characterization of particulates in biologics formulation continues to be challenging. The quantity of small size aggregates (e.g., dimer) in a stable biologics formulation is well controlled using precision analytical techniques (e.g., high-performance liquid chromatography). Particulate in clinical and commercial formulations is monitored using visual inspection and subvisible particulate counting assays. While visual inspection (by human eye or automated systems) is intended to detect particulates (intrinsic and extrinsic) of ~100 μm or larger, the subvisible counting methods cover smaller size ranges down to 10 μm. It is well recognized that research of particulates in the submicron (<1 μm) and low-micron (1–10 μm) ranges may provide important clues to understand the mechanism of particulate formation. The recent years have seen a significant increase in the development of newer technologies for more comprehensive characterization of particulates. This is attributed to increased awareness in this field of research over the past 5 years, stimulated by scholarly articles, commentaries, and robust discussions in various forums. This article provides an overview of emerging detection technologies that provide complementary characterization data encompassing a wider size range of particulates. It also discusses their advantages and limitations in the context of applications in biotherapeutics development.KEY WORDS: biotherapeutics, formulation development, laser diffraction, particulate matter, protein aggregation     

NONVERBAL TREATMENT OF NEUROSIS—Techniques for General Practice

“Psychosomatic medicine” does not demand that the general practitioner function as a psychiatrist; rather, it is a psychiatric orientation that can increase the effectiveness of purely medical treatment for such conditions as neuroses. The general practitioner to whom the patient turns may achieve permanent results with nonverbal techniques where formal psychotherapy would be impracticable or unacceptable.The first aim is to relieve pressure so that the patient can regain his mental balance and thereby his self-confidence. Arts, hobbies, sports, and the like can be prescribed rather specifically according to the patient''s personality and needs. Nutrition can be improved simply at first by prescribing needed additions to diet rather than imposing restrictions. Vitamin deficiency may by itself be the cause of neurosis or more serious mental disease, whereas psychic stress by itself may create a need for additional vitamin intake. Hormone therapy may be extremely helpful but must be based on clear indication and limited to specific purposes.Since lack of sleep and rest quickly impairs mental function, it is important for neurotic persons to learn relaxation as a necessity for sleep. Sedatives may be used in a crisis but should be abandoned as soon as possible.With all drugs there are problems of excess and habituation. The least, the mildest, the shortest dosage is the ideal.The initial steps of psychotherapy are available to any physician: Establishing rapport, noting how complaints are stated, encouraging ventilation, winning confidence rather than immediate results.     

A New Amylose Derivative for the Preparation of Protein–Carbohydrate Conjugates

N,N-Dimethylformamidase (DMFase) from Alcaligenes sp. strain KUFA-1, a bacterium that can grow on N,N-dimethylformamide (DMF) as the sole carbon and nitrogen source, catalyzes the first step of the DMF degradation. The DMFase gene dmfA1A2 was cloned in Escherichia coli, and its nucleotides were sequenced. The deduced amino acid sequence of the enzyme consisted of two α- and two β-subunits with 132 and 762 amino acids, respectively, and had little similarity to sequences in protein databases, including various amidases. The protein may be a new kind of amidase. DMFase activity was detected in E. coli cells transformed with an expression plasmid of the cloned DMFase gene. The properties of recombinant DMFase purified from E. coli were identical to those of Alcaligenes DMFase.     

New challenge for bionics—brain-inspired computing

By definition,bionics is the application of biological mechanisms found in nature to artificial systems in order to achieve specific functional goals.Successful examples range from Velcro,the touch fastener inspired by the hooks of burrs,to self-cleaning material,inspired by the surface of the lotus leaf.Recently,a new trend in bionics—Brain-Inspired Computing (BIC)-has captured increasing attention.Instead of learning from burrs and leaves,BIC aims to understand the brain and then utilize its operating principles to achieve powerful and efficient information processing.     

Acetone Precipitation—An Improved Procedure for the Isolation of Soybean Agglutinin

Isolation of soybean agglutinin (SBA) by the salt fractionation involves excessive amounts of (NH₄) ₂ SO ₄. We have found that SBA could be fractionally precipitated from an aqueous extract by adding acetone (40% final concentration). It is stable under these conditions for minimum 2 h at 5°C and 25°C. Incorporating these results, an improved procedure for the isolation of SBA has been developed. The SBA isolated by this method is obtained in better yield, has 6000 HU/mg protein and is identical to that isolated by the (NH₄)₂ SO ₄ method as ascertained by chromatographic and electro-phoretic comparisons and hapten inhibition assays.     

A New Paradigm for Enzymatic Control of α-Cleavage and β-Cleavage of the Prion Protein

The cellular form of the prion protein (PrP^C) is found in both full-length and several different cleaved forms in vivo. Although the precise functions of the PrP^C proteolytic products are not known, cleavage between the unstructured N-terminal domain and the structured C-terminal domain at Lys-109↓His-110 (mouse sequence), termed α-cleavage, has been shown to produce the anti-apoptotic N1 and the scrapie-resistant C1 peptide fragments. β-Cleavage, residing adjacent to the octarepeat domain and N-terminal to the α-cleavage site, is thought to arise from the action of reactive oxygen species produced from redox cycling of coordinated copper. We sought to elucidate the role of key members of the ADAM (a disintegrin and metalloproteinase) enzyme family, as well as Cu²⁺ redox cycling, in recombinant mouse PrP (MoPrP) cleavage through LC/MS analysis. Our findings show that although Cu²⁺ redox-generated reactive oxygen species do produce fragmentation corresponding to β-cleavage, ADAM8 also cleaves MoPrP in the octarepeat domain in a Cu²⁺- and Zn²⁺-dependent manner. Additional cleavage by ADAM8 was observed at the previously proposed location of α-cleavage, Lys-109↓His-110 (MoPrP sequencing); however, upon addition of Cu²⁺, the location of α-cleavage shifted by several amino acids toward the C terminus. ADAM10 and ADAM17 have also been implicated in α-cleavage at Lys-109↓His-110; however, we observed that they instead cleaved MoPrP at a novel location, Ala-119↓Val-120, with additional cleavage by ADAM10 at Gly-227↓Arg-228 near the C terminus. Together, our results show that MoPrP cleavage is far more complex than previously thought and suggest a mechanism by which PrP^C fragmentation responds to Cu²⁺ and Zn²⁺.     

Polar or Apolar—The Role of Polarity for Urea-Induced Protein Denaturation

Urea-induced protein denaturation is widely used to study protein folding and stability; however, the molecular mechanism and driving forces of this process are not yet fully understood. In particular, it is unclear whether either hydrophobic or polar interactions between urea molecules and residues at the protein surface drive denaturation. To address this question, here, many molecular dynamics simulations totalling ca. 7 µs of the CI2 protein in aqueous solution served to perform a computational thought experiment, in which we varied the polarity of urea. For apolar driving forces, hypopolar urea should show increased denaturation power; for polar driving forces, hyperpolar urea should be the stronger denaturant. Indeed, protein unfolding was observed in all simulations with decreased urea polarity. Hyperpolar urea, in contrast, turned out to stabilize the native state. Moreover, the differential interaction preferences between urea and the 20 amino acids turned out to be enhanced for hypopolar urea and suppressed (or even inverted) for hyperpolar urea. These results strongly suggest that apolar urea–protein interactions, and not polar interactions, are the dominant driving force for denaturation. Further, the observed interactions provide a detailed picture of the underlying molecular driving forces. Our simulations finally allowed characterization of CI2 unfolding pathways. Unfolding proceeds sequentially with alternating loss of secondary or tertiary structure. After the transition state, unfolding pathways show large structural heterogeneity.     

Efficient Acetone–Butanol–Ethanol Production from Corncob with a New Pretreatment Technology—Wet Disk Milling

Acetone–butanol–ethanol (ABE) production from corncob was achieved using an integrated process combining wet disk milling (WDM) pretreatment with enzymatic hydrolysis and fermentation by Clostridium acetobutylicum SE-1. Sugar yields of 71.3 % for glucose and 39.1 % for xylose from pretreated corncob were observed after enzymatic hydrolysis. The relationship between sugar yields and particle size of the pretreated corncob was investigated, suggesting a smaller particle size benefits enzymatic hydrolysis with the WDM pretreatment approach. Analysis of the correlation between parameters representing particle size and efficiency of enzymatic hydrolysis predicted that frequency 90 % is the best parameter representing particle size for the indication of the readiness of the material for enzymatic hydrolysis. ABE production from corncob was carried out with both separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes using C. acetobutylicum SE-1. Interestingly, when considering the time for fermentation as the time for ABE production, a comparable rate of sugar consumption and ABE production in the SHF process (0.55 g/l·h sugar consumption and 0.20 g/l·h ABE production) could be observed when glucose (0.50 g/l·h sugar consumption and 0.17 g/l·h ABE production) or a mixture of glucose and xylose (0.68 g/l·h sugar consumption and 0.22 g/l·h ABE production) mimicking the corncob hydrolysate was used as the substrate for fermentation. This result suggested that the WDM is a suitable pretreatment method for ABE production from corncob owing to the mild conditions. A higher ABE production rate could be observed with the SSF process (0.15 g/l·h) comparing with SHF process (0.12 g/l·h) when combining the time for saccharification and fermentation and consider it as the time for ABE production. This is possibly a result of low sustained sugar level during fermentation. These investigations lead to the suggestion that this new WDM pretreatment method has the potentials to be exploited for efficient ABE production from corncob.     

PLASMANATE?—A New Plasma Substitute for Pediatric Therapy

Plasmanate®, a human-serum protein solution, appears to have all the attributes of an ideal plasma expander. Freedom from infection, immediate availability in a clear, stable solution and the apparent absence of antigenic properties are particularly valuable qualities. The efficacy and safety of Plasmanate was clinically demonstrated in the treatment of 125 infants and children. This solution seems especially effective in the treatment of acute shock states and for the physiologic correction of hypoproteinemia. Comparison with other plasma expanders makes Plasmanate the agent of choice in the initial treatment of shock states in pediatrics.     

Precautionary Defaults—A New Strategy for Chemical Risk Management

In order to give adequate support to risk managers, new risk assessment methods should be developed that are (1) scientifically sound, (2) simplified, and (3) suited for precautionary risk management. In this Perspective we propose that the notion of a precautionary default can be a useful tool in the development of such methods. A precautionary default is a cautious or pessimistic assumption that is used in the absence of adequate information and that should be replaced when such information is obtained. Furthermore, we point out some promising research areas for the development of such indicators, viz. connections between chemical characteristics such as persistence and effect parameters, monitoring of contaminants in polar regions, monitoring of contaminants in breast milk, application of results from (human) toxicology in ecotoxicology and vice versa, (eco)toxicological test systems that are sensitive to effects on reproduction, and the application of bioinformatic methods to complex data, both in genomic research and in ecotoxicology. We conclude that precautionary decision-making does not require less science, but to the contrary it requires more science and improved communication between scientists and risk managers.     

Personalized Method for Interpreting Dreams (PMID)—As Applied to Relationship Issues

The purpose of this study was to develop a dreamwork model that would help individuals deal with relationship issues. Seventy dreams, involving seven major relationships, were selected from the woman participant's dreams. A dream interpretation model, the Personalized Method for Interpreting Dreams (PMID) was developed. Well-founded concepts in the PMID are: 1) dreams reflect emotions; and, 2) pre-dream thoughts, current circumstances, and personal definitions build dream meanings. The newest dreamwork concept of the PMID is the systemic perspective that relationship issues are best understood by discovering how relationship experiences influence our thoughts, emotions and behavior in other relationships. With a dreamwork systemic approach, the individual gathers together and studies series of dreams about major relationships in his or her life, primarily the family. Results of the thesis study show that the participant's use of the model was a factor in reducing stressful relationship issues.     

Two Distinct Mechanisms for Actin Capping Protein Regulation—Steric and Allosteric Inhibition

The actin capping protein (CP) tightly binds to the barbed end of actin filaments, thus playing a key role in actin-based lamellipodial dynamics. V-1 and CARMIL proteins directly bind to CP and inhibit the filament capping activity of CP. V-1 completely inhibits CP from interacting with the barbed end, whereas CARMIL proteins act on the barbed end-bound CP and facilitate its dissociation from the filament (called uncapping activity). Previous studies have revealed the striking functional differences between the two regulators. However, the molecular mechanisms describing how these proteins inhibit CP remains poorly understood. Here we present the crystal structures of CP complexed with V-1 and with peptides derived from the CP-binding motif of CARMIL proteins (CARMIL, CD2AP, and CKIP-1). V-1 directly interacts with the primary actin binding surface of CP, the C-terminal region of the α-subunit. Unexpectedly, the structures clearly revealed the conformational flexibility of CP, which can be attributed to a twisting movement between the two domains. CARMIL peptides in an extended conformation interact simultaneously with the two CP domains. In contrast to V-1, the peptides do not directly compete with the barbed end for the binding surface on CP. Biochemical assays revealed that the peptides suppress the interaction between CP and V-1, despite the two inhibitors not competing for the same binding site on CP. Furthermore, a computational analysis using the elastic network model indicates that the interaction of the peptides alters the intrinsic fluctuations of CP. Our results demonstrate that V-1 completely sequesters CP from the barbed end by simple steric hindrance. By contrast, CARMIL proteins allosterically inhibit CP, which appears to be a prerequisite for the uncapping activity. Our data suggest that CARMIL proteins down-regulate CP by affecting its conformational dynamics. This conceptually new mechanism of CP inhibition provides a structural basis for the regulation of the barbed end elongation in cells.     

Emerging Law of the Sea Issues in the Antarctic Maritime Area: A Heritage for the New Century?

This article focuses on three emerging law of the sea issues for states cooperating in management of the Antarctic and its maritime area. The first of these is no newcomer: How to regulate the dramatic increase in illegal, unregulated and unreported fishing of Patagonian toothfish (Dissostichus eleginoides) in the Southern Ocean? The second question, according to the letter of the UN Convention on the Law of the Sea, awaits the countries claiming sovereignty over portions of territory in the Antarctic 10 years from the entry into force of the Convention for each of them. The question here is what to do with the requirement contained in that Convention relating to the submission of information on the outer limit of the continental shelf beyond 200 nautical miles to the Commission on the Continental Shelf? Finally, there is a third tricky question: Who is competent to regulate, and accordingly to ban, mineral activities in the Southern Ocean seabed? Is it the International Seabed Authority as the global body, or the Antarctic Treaty Consultative Parties through their regional cooperation? This question may well never be put on the policy agenda for any global forum; but it may well be posed at any time and by any third party, whether in the UN General Assembly or, more likely, in the Assembly of the International Seabed Authority.     

Synonymous Codon Usage—a Guide for Co-Translational Protein Folding in the Cell

Molecular Biology - Abstract—In the cell, protein folding begins during protein synthesis/translation and thus is a co-translational process. Co-translational protein folding is tightly...     

Beyond Transcription—New Mechanisms for the Regulation of Molecular Chaperones

Molecular chaperones are an essential part of the universal heat shock response that allows organisms to survive stress conditions that cause intracellular protein unfolding. During the past few years, two new mechanisms have been found to control the activity of several chaperones under stress conditions—the regulation of chaperone activity by the redox state and by the temperature of the environment. Hsp33, for example, is redox-regulated. Hsp33 is specifically activated by disulfide bond formation during oxidative stress, where it becomes a highly efficient chaperone holdase that binds tightly to unfolding proteins. Certain small heat shock proteins, such as Hsp26 and Hsp16.9, on the other hand, are temperature regulated. Exposure to heat shock temperatures causes these oligomeric proteins to disassemble, thereby changing them into highly efficient chaperones. The ATP-dependent chaperone folding system DnaK/DnaJ/GrpE also appears to be temperature regulated, switching from a folding to a holding mode during heat stress. Both of these novel post-translational regulatory strategies appear to have one ultimate goal: to significantly increase the substrate binding affinity of the affected chaperones under exactly those stress conditions that require their highest chaperone activity. This ensures that protein folding intermediates remain bound to the chaperones under stress conditions and are released only after the cells return to non-stress conditions.     

Bioinformatics Goes to School—New Avenues for Teaching Contemporary Biology

Since 2010, the European Molecular Biology Laboratory''s (EMBL) Heidelberg laboratory and the European Bioinformatics Institute (EMBL-EBI) have jointly run bioinformatics training courses developed specifically for secondary school science teachers within Europe and EMBL member states. These courses focus on introducing bioinformatics, databases, and data-intensive biology, allowing participants to explore resources and providing classroom-ready materials to support them in sharing this new knowledge with their students.In this article, we chart our progress made in creating and running three bioinformatics training courses, including how the course resources are received by participants and how these, and bioinformatics in general, are subsequently used in the classroom. We assess the strengths and challenges of our approach, and share what we have learned through our interactions with European science teachers.     

The Latex Serologic Test for Syphilis—A New Screening Procedure

A new macroscopic screening test for syphilis, the Latex-sts test, is extraordinarily simple. After inactivation of the patient''s serum for 30 minutes at 56°C the test is performed by mixing the patient''s serum with latex particles coated with cardiolipin and a protein fraction obtained from the non-pathogenic Reiter strain of Treponema pallidum. Two to three minutes after mixing, the result of the test is observed on a ringed serologic plate. The sensitivity, specificity and reproducibility of the new test are equivalent to those of the qualitative Venereal Disease Research Laboratory tube test. The advantages of the Latex-sts are that it can be done in a short time, it is simple and it requires a minimum of laboratory equipment. The coated latex particles are stable for 12 months.     

N-glycosylation in Archaea—New roles for an ancient posttranslational modification

Genome analysis points to N-glycosylation as being an almost universal posttranslational modification in Archaea. Although such predictions have been confirmed in only a limited number of species, such studies are making it increasingly clear that the N-linked glycans which decorate archaeal glycoproteins present diversity in terms of both glycan composition and architecture far beyond what is seen in the other two domains of life. In addition to continuing to decipher pathways of N-glycosylation, recent efforts have revealed how Archaea exploit this variability in novel roles. As well as encouraging glycoprotein synthesis, folding and assembly into properly functioning higher ordered complexes, N-glycosylation also provides Archaea with a strategy to cope with changing environments. Archaea can, moreover, exploit the apparent species-specific nature of N-glycosylation for selectivity in mating, and hence, to maintain species boundaries, and in other events where cell-selective interactions are required. At the same time, addressing components of N-glycosylation pathways across archaeal phylogeny offers support for the concept of an archaeal origin for eukaryotes. In this MicroReview, these and other recent discoveries related to N-glycosylation in Archaea are considered.