Antibody drug conjugates of cleavable amino-alkyl and aryl maytansinoids

Natural products have been used for many medicinal purposes for centuries. Antibody drug conjugates (ADCs) have utilized this rich source of small molecule therapeutics to produce several clinically useful treatments. ADCs based on the natural product maytansine have been successful clinically. The authors further the utility of the anti-cancer natural product maytansine by developing efficacious payloads and linker-payloads for conjugating to antibodies. The success of our approach was realized in the EGFRvIII targeting ADC EGFRvIII-16. The ADC was able to regress tumors in 2 tumor models (U251/EGFRvIII and MMT/EGFRvIII). When compared to a positive control ADC, the efficacy observed was similar or improved while the isotype control ADCs had no effect.     

Spatiotemporal and demographic variation in the diet of New Zealand lesser short‐tailed bats (Mystacina tuberculata)

Variation in the diet of generalist insectivores can be affected by site‐specific traits including weather, habitat, and season, as well as demographic traits such as reproductive status and age. We used molecular methods to compare diets of three distinct New Zealand populations of lesser short‐tailed bats, Mystacina tuberculata. Summer diets were compared between a southern cold‐temperate (Eglinton) and a northern population (Puroera). Winter diets were compared between Pureora and a subtropical offshore island population (Hauturu). This also permitted seasonal diet comparisons within the Pureora population. Lepidoptera and Diptera accounted for >80% of MOTUs identified from fecal matter at each site/season. The proportion of orders represented within prey and the Simpson diversity index, differed between sites and seasons within the Pureora population. For the Pureora population, the value of the Simpson diversity index was higher in summer than winter and was higher in Pureora compared to Eglinton. Summer Eglinton samples revealed that juvenile diets appeared to be more diverse than other demographic groups. Lactating females had the lowest dietary diversity during summer in Pureora. In Hauturu, we found a significant negative relationship between mean ambient temperature and prey richness. Our data suggest that M. tuberculata incorporate a narrower diversity of terrestrial insects than previously reported. This provides novel insights into foraging behavior and ecological interactions within different habitats. Our study is the first from the Southern Hemisphere to use molecular techniques to examine spatiotemporal variation in the diet of a generalist insectivore that inhabits a contiguous range with several habitat types and climates.     

Extracellular Neutrophil Proteases Are Efficient Regulators of IL-1, IL-33, and IL-36 Cytokine Activity but Poor Effectors of Microbial Killing

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Trichloroethylene biodegradation by mesophilic and psychrophilic ammonia oxidizers and methanotrophs in groundwater microcosms.

This study investigated the efficiency of methane and ammonium for stimulating trichloroethylene (TCE) biodegradation in groundwater microcosms (flasks and batch exchange columns) at a psychrophilic temperature (12 degrees C) typical of shallow aquifers in the northern United States or a mesophilic temperature (24 degrees C) representative of most laboratory experiments. After 140 days, TCE biodegradation rates by ammonia oxidizers and methanotrophs in mesophilic flask microcosms were similar (8 to 10 nmol day-1), but [14C]TCE mineralization (biodegradation to 14CO2) by ammonia oxidizers was significantly greater than that by methanotrophs (63 versus 53%). Under psychrophilic conditions, [14C]TCE mineralization in flask systems by ammonia oxidizers and methanotrophs was reduced to 12 and 5%, respectively. In mesophilic batch exchange columns, average TCE biodegradation rates for methanotrophs (900 nmol liter-1 day-1) were not significantly different from those of ammonia oxidizers (775 nmol liter-1 day-1). Psychrophilic TCE biodegradation rates in the columns were similar with both biostimulants and averaged 145 nmol liter-1 day-1. Methanotroph biostimulation was most adversely affected by low temperatures. At 12 degrees C, the biodegradation efficiencies (TCE degradation normalized to microbial activity) of methanotrophs and ammonia oxidizers decreased by factors of 2.6 and 1.6, respectively, relative to their biodegradation efficiencies at 24 degrees C. Collectively, these experiments demonstrated that in situ bioremediation of TCE is feasible at the psychrophilic temperatures common in surficial aquifers in the northern United States and that for such applications biostimulation of ammonia oxidizers could be more effective than has been previously reported.     

Induction of aquaporin-1 mRNA following cardiopulmonary bypass and reperfusion.

BACKGROUND: Cardiopulmonary bypass (CPB) and hypothermic circulatory arrest (HCA) are important components of congenital cardiac surgery. Ischemia/reperfusion injury and inflammatory cascade activation result in endothelial damage and vascular leak which are clinically manifested as pulmonary edema and low cardiac output postoperatively. Newborns are particularly susceptible. Subtraction cloning is a useful method of isolating induced genes and can be applied to CPB/HCA. MATERIALS AND METHODS: We used a newborn lamb model replicating infant CPB with HCA to obtain tissues during various periods of reperfusion. We utilized subtraction cloning to identify mRNA induced in lung following CPB/HCA and reperfusion. Ribonuclease protection was used to quantify mRNA levels. RESULTS: We isolated a cDNA encoding ovine aquaporin-1 in a subtracted cDNA screen comparing control lung with lung exposed to CPB/HCA and reperfusion. Aquaporin-1 mRNA levels increased 3-fold in lung (p = .006) exposed to CPB/HCA and 6 hr of reperfusion. No induction was observed immediately following bypass or after 3 hr of reperfusion. We found no significant induction of aquaporin-1 mRNA following bypass, arrest, and reperfusion in other tissues surveyed, including ventricle, atrium, skeletal muscle, kidney, brain, and liver. CONCLUSIONS: Our finding that aquaporin-1 mRNA is reproducibly induced in lung following CPB/HCA with 6 hr of reperfusion suggests an important role for the water channel in the setting of pulmonary edema. Induction of Aquaporin-1 is late compared with other inflammatory mediators (ICAM-1, E-selectin, IL-8). Further studies are needed to determine if aquaporin-1 contributes to the disease process or if it is part of the recovery phase.     

Temperature and salts effects on the peptidase activities of the recombinant metallooligopeptidases neurolysin and thimet oligopeptidase.

We report the recombinant neurolysin and thimet oligopeptidase (TOP) hydrolytic activities towards internally quenched fluorescent peptides derived from the peptide Abz-GGFLRRXQ-EDDnp (Abz, ortho-aminobenzoicacid; EDDnp, N-(2,4-dinitrophenyl) ethylenediamine), in which X was substituted by 11 different natural amino acids. Neurolysin hydrolyzed these peptides at R-R or at R-X bonds, and TOP hydrolyzed at R-R or L-R bonds, showing a preference to cleave at three or four amino acids from the C-terminal end. The kinetic parameters of hydrolysis and the variations of the cleavage sites were evaluated under different conditions of temperature and salt concentration. The relative amount of cleavage varied with the nature of the substitution at the X position as well as with temperature and NaCl concentration. TOP was activated by all assayed salts in the range 0.05-0.2 m for NaCl, KCl, NH4Cl and NaI, and 0.025-0.1 m for Na2SO4. Concentration higher than 0.2 N NH4Cl and NaI reduced TOP activity, while 0.5 N or higher concentration of NaCl, KCl and Na2SO4 increased TOP activity. Neurolysin was strongly activated by NaCl, KCl and Na2SO4, while NH4Cl and NaI have very modest effect. High positive values of enthalpy (DeltaH*) and entropy (DeltaS*) of activation were found together with an unusual temperature dependence upon the hydrolysis of the substrates. The effects of low temperature and high NaCl concentration on the hydrolytic activities of neurolysin and TOP do not seem to be a consequence of large secondary structure variation of the proteins, as indicated by the far-UV CD spectra. However, the modulation of the activities of the two oligopeptidases could be related to variations of conformation, in limited regions of the peptidases, enough to modify their activities.     

TAXONOMIC STATUS AND GEOGRAPHICAL CRANIAL VARIATION OF COMMON DOLPHINS (DELPHINUS) IN THE EASTERN NORTH ATLANTIC

The common dolphin has a widespread distribution and is relatively abundant in the temperate to subtropical waters of the eastern North Atlantic. However, it is not known whether different species, subspecies, or populations occur in this region. We examined 393 common dolphin skulls obtained from both stranded and bycaught individuals collected between 1901 and 2005. The series included skulls of 152 females and 199 males, from animals ranging in body length from 93 to 230 cm and 105 to 244 cm, respectively. The ranges of total body length, skull size, RL/ZGW ratio and maximum upper alveolar (tooth) count of common dolphins in the eastern North Atlantic overlapped with those of both short- ( D. delphis ) and long-beaked ( D. capensis ) species found off the Californian coast. However, in the absence of additional data, the common dolphin in the eastern North Atlantic is regarded here as a large form of Delphinus delphis . Sexual dimorphism and possible sex-linked characters were identified within the sample. Results of the current study indicate some population differentiation within the eastern North Atlantic, with common dolphins off Portugal showing segregation in morphometric characteristics from common dolphins in other areas.     

External morphology of the short-beaked common dolphin, Delphinus delphis: growth, allometric relationships and sexual dimorphism

Growth, allometric relationships and sexual dimorphism are described from measurements of 105 male, 149 female and 38 unsexed specimens of short‐beaked common dolphin, Delphinus delphis, stranded along the Irish coastline (53.8% of the sample) or by‐caught in fisheries (46.2% of the sample), from 1990 to 2003. For each dolphin, 24 external body length measurements were recorded. Ages were determined for 183 dolphins by analysis of growth layer groups in the dentine. Males ranged in total body length (TBL) from 105 to 231 cm and females from 93 to 230 cm, with a maximum age of 25 years obtained for both sexes. Using a single Gompertz growth curve, asymptotic values obtained for TBL were 211.6 cm and 197.4 cm for males and females, respectively. Asymptotic lengths were attained at 11 years in males and 9 years in females. The gestation period was estimated to last approximately 11.5 months. Sexual size dimorphism (SSD) was evident, with males being significantly larger than females for 20 of the characters measured, and an SSD ratio of 1.06 was obtained. Sexual shape dimorphism was lacking, except for the presence of prominent postanal humps in mature males.     

Long-Term Evolution of the Hypervariable Region of Hepatitis C Virus in a Common-Source-Infected Cohort

The long-term evolution of the hepatitis C virus hypervariable region (HVR) and flanking regions of the E1 and E2 envelope proteins have been studied in a cohort of women infected from a common source of anti-D immunoglobulin. Whereas virus sequences in the infectious source were relatively homogeneous, distinct HVR variants were observed in each anti-D recipient, indicating that this region can evolve in multiple directions from the same point. Where HVR variants with dissimilar sequences were present in a single individual, the frequency of synonymous substitution in the flanking regions suggested that the lineages diverged more than a decade previously. Even where a single major HVR variant was present in an infected individual, this lineage was usually several years old. Multiple lineages can therefore coexist during long periods of chronic infection without replacement. The characteristics of amino acid substitution in the HVR were not consistent with the random accumulation of mutations and imply that amino acid replacement in the HVR was strongly constrained. Another variable region of E2 centered on codon 60 shows similar constraints, while HVR2 was relatively unconstrained. Several of these features are difficult to explain if a neutralizing immune response against the HVR is the only selective force operating on E2. The impact of PCR artifacts such as nucleotide misincorporation and the shuffling of dissimilar templates is discussed.     

Behaviour of DNA,protein and acid hydrolases in response to thyroxine in isolated tail tips ofXenopus-larvae

Summary Analysis of factors influencing survival of tail tips ofXenopus larvae in vitro has shown that prevention of infection by antibiotic pretreatment of donor tadpoles and amputated tips is most critical. In tail tips exceeding not more than 1/3 of the body length, maintenance of N-content and regenerative capacity are superior in Niu-Twitty and Steinberg's saline than in Holtfreter's solution, all media being supplemented by 0.04 % sulfathiazole. Addition of nutrient ingredients to saline (glucose, Parker's medium 199, serum protein) does not improve viability of tail explants.In isolated tail tips 2.5×10^–7M L-thyroxine (T₄) induces involution in vitro, resulting in losses of about 85% in DNA and 70% of protein respectively after 12 days of treatment. A significant decrease in DNA occurs after three days, and for protein after 6 days of hormone treatment, when tail fins are almost fully resorbed.Statistical analysis of the regression curves for decrease in DNA and protein, produced by different concentrations of T₄, indicates the presence of an upper threshold (2.5×10^–7M) and a lower threshold (2.5×10^–8M) of sensitivity to hormone. Intermediate concentrations affect the latency time required for onset of DNA and protein loss, lengthening it at lower concentration.In tail tips exposed to 2.5×10^–7M T₄ a concurrent rise in activity of cathepsins, DNase and acid phosphatase has been demonstrated. The specific activities of these acid hydrolases are significantly higher in T₄-treated tails after four days of hormone administration, this response proceeding detectable loss in protein by two days. Extent and duration in the rise of activity are characteristic for each enzyme, the increase in both specific and total activities being highest for cathepsins, intermediate for DNase and lowest for acid phosphatase.

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Zusammenfassung Die Untersuchung der für das Überleben von isolierten Schwanzspitzen vonXenopus-Larven wesentlichen Kulturbedingungen hat ergeben, daß die Verhütung von Infektionen durch Vorbehandlung der Kaulquappen und der amputierten Schwanzspitzen für den Erfolg entscheidend ist. Schwanzspitzen, deren Länge 1/3 der Körperlänge der Kaulquappen nicht überschreitet, zeigen einen geringeren Verlust an N-haltigen Stoffen und ein besseres Regenerationsvermögen in Niu-Twitty- und Steinberglösung gegenüber der Lösung nach Holtfreter, obwohl alle Medien 0,04% Sulfathiazol enthalten. Zusatz von Nährstoffen (Glucose, Parker's Medium 199, Serumprotein) ergeben keine Verbesserung der Überlebensfähigkeit.In isolierten Schwanzspitzen bewirken 2,5×10^–7M L-Thyroxin (T₄) die Rückbildung in vitro, wobei im Verlaufe von 12 Tagen der DNS-Gehalt um 85% und derjenige an Protein um 70% sinkt. Der DNS-Verlust ist bereits nach 3 Tagen signifikant, während eine signifikante Abnahme an Protein erst nach 6 Tagen Hormonbehandlung nachgewiesen werden kann, wenn die Flossensäume bereits resorbiert sind.Aufgrund der statistischen Analyse der für den DNS- und Proteinverlust in Abhängigkeit von verschiedenen T₄-Konzentrationen ermittelten Regressionsgeraden ergeben sich folgende Aussagen: 2,5×10^–7 M repräsentiert die obere und 2,5×10^–8 M T₄ die untere Empfindlichkeitsschwelle. Innerhalb dieses Bereiches zeigt die Latenzzeit für den Beginn der DNS -und Proteinabnahme, nicht aber die Geschwindigkeit des Involutionsprozesses eine Konzentrationsabhängigkeit, indem die Latenzzeit mit fallender T₄-Konzentration zunimmt.Unter der Einwirkung von 2,5×10^–7 M T₄ nimmt in den Schwanzspitzen die Aktivität von Kathepsinen, DNase und saurer Phosphatase gleichzeitig zu. Die spezifische Aktivität dieser sauren Hydrolasen ist bereits nach 4 Tagen Hormonbehandlung signifikant erhöht, während ein signifikanter Proteinverlust erst am 6. Tag der Behandlung nachgewiesen werden kann. Ausmaß und Dauer der Aktivitätszunahme sind für jedes Enzym charakteristisch; die Zunahme der spezifischen und Gesamtaktivität ist am größten bei den Kathepsinen, etwas geringer bei der DNase und am niedrigsten bei der sauren Phosphatase.

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These studies were carried out within the scope of a project supported by the Schweizerischer Nationalfonds zur Förderung der wissenschaftlichen Forschung under the direction of Prof. B. Weber.

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The author would like to acknowledge the advice and encouragement of Prof. R. Weber in all phases of these studies, and the assistance of Prof. S. Rosin with the statistical techniques. Further, the technical assistance of Miss A. Rohner, Miss B. Fahrer, Mr. J. Zbären, and Mr. T. Wyler is gratefully acknowledged.