Metal ion interactions with mAbs: Part 1

Fragmentation in the hinge region of an IgG1 monoclonal antibody (mAb) can affect product stability, potentially causing changes in potency and efficacy. Metals ions, such as Cu²⁺, can bind to the mAb and undergo hydrolysis or oxidation, which can lead to cleavage of the molecule. To better understand the mechanism of Cu²⁺-mediated mAb fragmentation, hinge region cleavage products and their rates of formation were studied as a function of pH with and without Cu²⁺. More detailed analysis of the chemical changes was investigated using model linear and cyclic peptides (with the sequence of SCDKTHTC) derived from the upper hinge region of the mAb. Cu²⁺ mediated fragmentation was determined to be predominantly via a hydrolytic pathway in solution. The sites and products of hydrolytic cleavage are pH and strain dependent. In more acidic environments, rates of Cu²⁺ induced hinge fragmentation are significantly slower than at higher pH. Although the degradation reaction rates between the linear and cyclic peptides are not significantly different, the products of degradation vary. mAb fragmentation can be reduced by modifying His, which is a potential metal binding site and a known ligand in other metalloproteins. These results suggest that a charge may contribute to stabilization of a specific molecular structure involved in hydrolysis, leading to the possible formation of a copper binding pocket that causes increased susceptibility of the hinge region to degradation.     

Ant–scale mutualism increases scale infestation,decreases folivory,and disrupts biological control in restored tropical forests

Ant–hemipteran mutualisms can have positive and negative effects on host plants depending on the level of hemipteran infestation and plant protection conferred by ants against folivory. Differential effects of such mutualisms on plant survival are well documented in undisturbed and ant-invaded systems, but few have explored how anthropogenic disturbance affects interactions between hemipterans and native ant species and what the consequences may be for recovering ecosystems. Within a fragmented landscape in Costa Rica, restored tropical forests harbor a mutualism between the native ant Wasmannia auropunctata and the scale insect Alecanochiton marquesi on the abundant, early-successional tree Conostegia xalapensis. I added A. marquesi scales to C. xalapensis seedlings and either allowed or excluded W. auropunctata to investigate if this mutualism leads to increased scale infestation, decreased scale mortality, and decreased folivory. I also examined whether these effects are mediated by the percentage of remnant forest cover in the landscape. I found that seedlings with ants excluded had fewer scale insects and higher herbivory than plants with ants present. I also found evidence that scale mortality due to fungal attack and parasitism was higher on ant-excluded versus ant-allowed seedlings but only at sites with high surrounding landscape forest cover. Together, these results suggest that mutualisms between scale insects and native ants can promote scale infestation, reduce folivory on native plant species, and potentially disrupt biological control of scale insects in recovering tropical forests. Further, my experiment underscores the importance of remnant tropical forests as sources of biological control in anthropogenically disturbed landscapes. Abstract in Spanish is available with online material.     

The effect of two cryopreservation methods on human sperm DNA damage

Several methods are currently available for selection when conducting sperm cryopreservation, however, these methods might cause different degrees of damage on sperm DNA. The aim of the this study is to compare the effects of storage at −80 °C (in ultra-low temperature refrigerator) and at −196 °C (in liquid nitrogen) on sperm DNA damage, thus to provide a reference for choosing the right method according to different aims. We randomly collected 28 semen samples from college students of Chongqing city. The samples stored at −80 °C were neat semen samples and the samples stored at −196 °C were mixed with additional cryoprotectants. Each sample was subjected to two freezing-thawing cycles, and the sperm DNA damage levels of fresh and thawed samples were measured by single cell gel electrophoresis (SCGE) and sperm chromatin structure assay (SCSA). Both SCGE and SCSA assays showed cryopreservation induced significant damage to sperm DNA. However, storage at −196 °C lead to more severe damage to sperm DNA than storage at −80 °C measured by SCSA. Sperm DNA damage increased simultaneously with the higher frequency of freezing-thawing cycles. We concluded that storage of neat semen samples at −80 °C had milder damage to sperm DNA than storage at −196 °C mixed with cryoprotectants. To avoid additional sperm DNA damage, repeated freezing and thawing should be prevented.     

The Influence of Large Tree Density on Howler Monkey (Alouatta palliata mexicana) Presence in Very Small Rain Forest Fragments

The populations of the Mexican mantled howler monkey ( Alouatta palliata mexicana ) in the Los Tuxtlas region, Mexico, have declined drastically due to habitat loss and fragmentation. Nevertheless, several troops still inhabit very small and isolated rain forest fragments. We identified the main vegetation attributes that can favor the presence of howlers within 18 small (< 10-ha) fragments that did not differ significantly in size, shape, and isolation (nine occupied and nine unoccupied by howlers). We found that habitat quality ( i.e. , food resources and vegetation structure) affected howler incidence in small fragments. Particularly, the occupied fragments showed greater density of big trees (dbh > 60 cm), greater total basal area, greater basal area of persistent tree species, and greater basal area of top food species than the unoccupied fragments; suggesting that even for small fragments the loss of big trees and particularly the decrease in size class of the top food species can negatively affect howler distribution in highly fragmented landscapes. These findings could be used to establish foreground conservation areas for this critically endangered subspecies in fragmented landscapes of Los Tuxtlas.     

Reduction of IL-2 fragmentation during manufacturing of a novel immunocytokine by DoE process optimization

Interleukin-2 (IL-2) is a potent molecule in cancer therapy. Clinical application, however, is limited due to its strong side effects during the treatment. We developed an IL-2 variant (IL-2v) immunocytokine to circumvent the drawbacks of the current IL-2 therapy. During the production of the IL-2v immunocytokine in Chinese hamster ovary (CHO) cells, molecules with fragmented IL-2v and therefore reduced cytokine activity can be observed. To control product fragmentation different production process conditions were investigated. By shifting temperature or pH after the cell growth phase to lower values, fragmented species can be reduced from 10% to 12% to about 4%. However, with the adopted process conditions, the effective titer is decreased concomitantly. Moreover, fermentation length and inoculation cell density are parameters to adjust fragmentation and effective titer. A suitable method for efficient process optimization is the design of experiment approach. With this procedure, novel optimal values for temperature, pH value, harvest day, and inoculation cell densities were proposed and tested subsequently. In comparison to the former process, the improved process reduces fragmentation by 66% while keeping the effective titer comparable. In summary, these findings will help to control fragmentation in CHO production processes of different IL-2v or IL-2 containing therapeutic proteins.     

Apoptosis in Etiolated Wheat Seedlings: 1. Ultrastructure of the Apoptotic Cell

We studied the process of apoptosis in etiolated wheat (Triticum aestivum L.) seedlings. As a result, an integral pattern of the apoptotic plant cell ultrastructure was established. In the apoptotic cells of the coleoptile, we observed chromatin condensation and margination, an increased density and specific cytoplasm fragmentation accompanied by the appearance of unusual cytoplasmic vesicles containing subcellular organelles, mitochondria in particular, in the vacuoles.     

A Comparison of the Phyllostomid Bat Assemblages in Undisturbed Neotropical Forest and in Forest Fragments of a Slash‐and‐Burn Farming Mosaic in Petén,Guatemala1

Using mist nets, we compared phyllostomid bat ensembles of continuous mature forest in Tikal National Park, Guatemala, and of forest fragments in the nearby farming landscape. Of 20 species captured, 13 were shared between treatments, 4 were unique to continuous forest, and 3 were unique to forest fragments. Dominance–diversity curves were similar for the two treatments except that Sturnira lilium comprised 43 percent of captures in the forest fragments, resulting in greater dominance there. Capture rates (and presumably relative abundance) differed significantly between continuous forest and forest fragments, both in terms of species and feeding guilds. Sturnia lilium and Dermanura sp. were captured significantly more often in forest fragments than in continuous forest, whereas Artibeus jamaicensis, A. lituratus, and Centurio senex were taken significantly more often in continuous forest. Large frugivores accounted for a higher proportion of total captures in continuous forest than in forest fragments, whereas small frugivores showed the opposite pattern. By their abundances, Carollia perspicillata and S. lilium are indicators of forest disturbance. The relative abundances of large frugivores, which feed on large fruits of mature forest trees, and small frugivores, which feed on small‐fruited plants occurring in early succession, are an indicator of forest disturbance. Other groups, such as large insect‐ and vertebrate‐eating bats, because of their low capture rates, are impractical as indicators for rapid assessment of forest disturbance based on mist netting, but may prove especially vulnerable to forest fragmentation.     

Induction of apoptosis and c-myc in L1210 lymphocytic leukemia cells by adenosine

High concentrations of adenosine (Ado), when added to L1210 lymphocytic leukemia cells, resulted in apoptosis or programmed cell death. The apoptotic process was accompanied by distinct morphological changes including chromatin condensation and blebbing of plasma membranes. Extensive DNA fragmentation was correlated with Ado concentrations. Furthermore, apoptosis in these cells was preceded by an early but transient expression of c-myc proto-oncogene, and was not influenced by homocysteine thiolactone added to the cells. Since severe combined immunodeficiency (SCID) is associated with a deficiency of adenosine deaminase, leading to defects in both cellular and humoral immunity, Ado-induced apoptosis may thus be a contributing factor in the pathology of SCID.     

High levels of variation despite genetic fragmentation in populations of the endangered mountain pygmy-possum, Burramys parvus, in alpine Australia

In endangered mammals, levels of genetic variation are often low and this is accompanied by genetic divergence among populations. The mountain pygmy-possum (Burramys parvus) is an endangered marsupial restricted to the alpine region of Victoria and New South Wales, Australia. By scoring variation at eight microsatellite loci, we found that B. parvus populations exhibit high levels of genetic divergence and fall into three distinct groups from the northern, central and southern areas of the distribution of this species, consistent with previous assessments of mitochondrial DNA variation. F(ST) values between populations from these regions ranged from 0.19 to 0.54. Within the central area, there was further genetic fragmentation, and a linear association between genetic and geographical distance. This pattern is likely to reflect limited dispersal across barriers despite the fact that individual B. parvus can move several kilometres. Levels of genetic variation within populations were high with the exception of a southern population where there was evidence of inbreeding. From a conservation perspective, all three areas where B. parvus are found should be considered as separate gene pools; management of populations within these areas needs to take into account the low gene flow between populations, as well as threats posed by roads, resorts and other developments in the alpine region. The low genetic variability and inbreeding in the southern population is of particular concern given the high levels of variability in other B. parvus populations.