Thermostable designed ankyrin repeat proteins (DARPins) as building blocks for innovative drugs

Designed ankyrin repeat proteins (DARPins) are antibody mimetics with high and mostly unexplored potential in drug development. By using in silico analysis and a rationally guided Ala scanning, we identified position 17 of the N-terminal capping repeat to play a key role in overall protein thermostability. The melting temperature of a DARPin domain with a single full-consensus internal repeat was increased by 8 °C to 10 °C when Asp17 was replaced by Leu, Val, Ile, Met, Ala, or Thr. We then transferred the Asp17Leu mutation to various backgrounds, including clinically validated DARPin domains, such as the vascular endothelial growth factor-binding domain of the DARPin abicipar pegol. In all cases, these proteins showed improvements in the thermostability on the order of 8 °C to 16 °C, suggesting the replacement of Asp17 could be generically applicable to this drug class. Molecular dynamics simulations showed that the Asp17Leu mutation reduces electrostatic repulsion and improves van-der-Waals packing, rendering the DARPin domain less flexible and more stable. Interestingly, this beneficial Asp17Leu mutation is present in the N-terminal caps of three of the five DARPin domains of ensovibep, a SARS-CoV-2 entry inhibitor currently in clinical development, indicating this mutation could be partly responsible for the very high melting temperature (>90 °C) of this promising anti-COVID-19 drug. Overall, such N-terminal capping repeats with increased thermostability seem to be beneficial for the development of innovative drugs based on DARPins.     

Behavioural and chemical mechanisms behind a Mediterranean ant–ant association

1. Interspecific competition among ants is common, and so is competitive exclusion among dominant ant species. In contrast, specific associations between non‐parasitic ant species are rare, especially in the temperate zones. As an exception, the subordinate ant Camponotus lateralis frequently co‐occurs with the dominant Crematogaster scutellaris but rarely with other dominant ants. 2. This association is one of various associations between Camponotus and Crematogaster species across the world. However, the mechanisms behind these co‐occurences are largely unknown. 3. In the present study, we therefore investigated the association of Ca. lateralis and Cr. scutellaris. We studied the spatial association of the nests, interspecific aggression, both species' cuticular hydrocarbon profiles, and their propensity to follow the other species' pheromone trails. 4. Crematogaster scutellaris usually attacked and displaced the generally submissive Ca. lateralis, but was significantly less aggressive at jointly used trails. Camponotus nests were always in close proximity to Crematogaster nests. 5. The cuticular hydrocarbons of both species consisted of alkanes with chain lengths between C21 and C35. The two species had 25 hydrocarbons in common, including mono‐, di‐, and tetramethyl alkanes. Despite this qualitative similarity, however, the quantitative hydrocarbon composition differed between the two species. 6. Camponotus lateralis followed artificial trails containing trail pheromones of Cr. scutellaris, but the latter did not follow Ca. lateralis trail pheromones. Interspecific trail‐following by Camponotus, but not vice versa, has been observed in another Camponotus–Crematogaster association and may be a more general mechanism that facilitates associations between the two ant genera.     

Thidiazuron-induced shoot regeneration in pigeonpea (Cajanus cajan L.)

Thidiazuron either alone or in combination with IAA induced high frequency shoot regeneration from primary leaf segments of three pigeonpea cultivars. Transfer of the cultures to medium with reduced concentration of thidiazuron resulted in further development of the shoots. The regenerated shoots were subsequently transferred to medium supplemented with BA, IAA and gibberellic acid where 5-10% of the shoots elongated further. Rooting of shoots could be obtained on half strength MS medium supplemented with NAA. Histological studies confirmed the mode of regeneration as shoot organogenesis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Opposing Effects of Low Molecular Weight Heparins on the Release of Inflammatory Cytokines from Peripheral Blood Mononuclear Cells of Asthmatics

Background

T-cell-mediated inflammatory cytokines, such as interleukin (IL)-4, IL-5, IL-13 and tumor necrosis factor-alpha (TNF-α), play an important role in the initiation and progression of inflammatory airways diseases. Low-molecular-weight heparins (LMWHs), widely used anticoagulants, possess anti-inflammatory properties making them potential treatment options for inflammatory diseases, including asthma. In the current study, we investigated the modulating effects of two LMWHs (enoxaparin and dalteparin) on the release of cytokines from stimulated peripheral blood mononuclear cells (PBMCs) of asthmatic subjects to identify the specific components responsible for the effects.

Methods

PBMCs from asthmatic subjects (consist of ~75% of T-cells) were isolated from blood taken from ten asthmatic subjects. The PBMCs were pre-treated in the presence or absence of different concentrations of LMWHs, and were then stimulated by phytohaemagglutinin for the release of IL-4, IL-5, IL-13 and TNF-α. LMWHs were completely or selectively desulfated and their anticoagulant effect, as well as the ability to modulate cytokine release, was determined. LMWHs were chromatographically fractionated and each fraction was tested for molecular weight determination along with an assessment of anticoagulant potency and effect on cytokine release.

Results

Enoxaparin inhibited cytokine release by more than 48%, whereas dalteparin increased their release by more than 25%. The observed anti-inflammatory effects of enoxaparin were independent of their anticoagulant activities. Smaller fractions, in particular dp4 (four saccharide units), were responsible for the inhibitory effect of enoxaparin. Whereas, the larger fractions, in particular dp22 (twenty two saccharide units), were associated with the stimulatory effect of dalteparin.

Conclusion

Enoxaparin and dalteparin demonstrated opposing effects on inflammatory markers. These observed effects could be due to the presence of structurally different components in the two LMWHs arising from different methods of depolymerisation. This study provides a platform for further studies investigating the usefulness of enoxaparin in various inflammatory diseases.     

Microwave treatment induced mutations and altered gene expression in <Emphasis Type="Italic">Vigna aconitifolia</Emphasis>

Primary leaf explants of aseptically grown seedlings of moth bean [Vigna aconitifolia (Jacq.) Marechal] immersed in water or not were treated in microwave oven (2450 MHz, 800 W cm⁻²) for 1, 3, 5 and 7 s before culturing. Callusing and shoot emergence from these explants were enhanced up to microwave exposure lasted 5 s while longer treatment of water-immersed explants delayed callusing. One polypeptide (26.6 kD) was up regulated in the callus derived from microwave treatment in water-immersed explants. RAPD analysis detected alteration in DNA sequences due to microwave treatment in water-immersed explants for 7 s. The frequency of mutation was 1.6 % (4 bands out of 248) over all the cultures analyzed and the same was 13 % (4 bands out of 31), if amplicons generated at 7 s treatment alone were considered.     

Prospects of genetic engineering of plants for phytoremediation of toxic metals

Bioremediation is gaining a lot of importance in recent times as an alternate technology for removal of elemental pollutants in soil and water, which require effective methods of decontamination. Phytoremediation--the use of green plants to remove, contain or render harmless environmental pollutants--may offer an effective, environmentally nondestructive and cheap remediation method. The use of genetic engineering to modify plants for metal uptake, transport and sequestration may open up new avenues for enhancing efficiency of phytoremediation. Metal chelator, metal transporter, metallothionein (MT), and phytochelatin (PC) genes have been transferred to plants for improved metal uptake and sequestration. Transgenic plants, which detoxify/accumulate cadmium, lead, mercury, arsenic and selenium have been developed. A better understanding of the mechanisms of rhizosphere interaction, uptake, transport and sequestration of metals in hyperaccumulator plants will lead to designing novel transgenic plants with improved remediation traits. As more genes related to metal metabolism are discovered, facilitated by the genome sequencing projects, new vistas will be opened up for development of efficient transgenic plants for phytoremediation.     

Tropical soil microflora of spice-based cropping systems as potential antagonists of root-knot nematodes

Suppression of plant parasitic nematodes with nematode predators, parasites or antagonists is an eco-friendly approach than the toxic chemicals. In a study, soil borne fungi from the rhizosphere of major spice crops were collected from diverse cropping systems prevailing in three southern states of India. A series of in vitro studies were conducted using 73 freshly collected fungal isolates and 76 isolates obtained from other sources. Out of this 67 isolates were not parasitic on females of root-knot nematodes whereas 115 isolates, though colonized the egg masses, did not show any signs of parasitism on nematode eggs. Fifty-nine isolates showed 50-90% inhibition in egg hatch. Pochonia chlamydospora, Verticillium lecanii, Paecilomyces lilacinus, and few isolates of Trichoderma spp. showed >25% parasitism on root-knot nematode eggs. The most promising isolates in this study were one isolate each of Aspergillus (F.45), Fusarium (F.47), and Penicillium (F.59); three each isolates of Trichoderma (F.3, F.52, and F.60) and Pochonia (F.30 and Vc.3) Verticillium (Vl); and two isolates of fungi that could not be identified (F.28 and F.62). Parasitism by Aspergillus tamarii, Aspergillus ustus, Drechslera sp., Humicola sp., and Scopulariopsis sp. on root-knot nematode eggs or females, reported in the present study, are new reports.     

Intra‐floral resource partitioning between endemic and invasive flower visitors: consequences for pollinator effectiveness

1. Sympatric flower visitor species often partition nectar and pollen and thus affect each other's foraging pattern. Consequently, their pollination service may also be influenced by the presence of other flower visiting species. Ants are solely interested in nectar and frequent flower visitors of some plant species but usually provide no pollination service. Obligate flower visitors such as bees depend on both nectar and pollen and are often more effective pollinators. 2. In Hawaii, we studied the complex interactions between flowers of the endemic tree Metrosideros polymorpha (Myrtaceae) and both, endemic and introduced flower‐visiting insects. The former main‐pollinators of M. polymorpha were birds, which, however, became rare. We evaluated the pollinator effectiveness of endemic and invasive bees and whether it is affected by the type of resource collected and the presence of ants on flowers. 3. Ants were dominant nectar‐consumers that mostly depleted the nectar of visited inflorescences. Accordingly, the visitation frequency, duration, and consequently the pollinator effectiveness of nectar‐foraging honeybees (Apis mellifera) strongly decreased on ant‐visited flowers, whereas pollen‐collecting bees remained largely unaffected by ants. Overall, endemic bees (Hylaeus spp.) were ineffective pollinators. 4. The average net effect of ants on pollination of M. polymorpha was neutral, corresponding to a similar fruit set of ant‐visited and ant‐free inflorescences. 5. Our results suggest that invasive social hymenopterans that often have negative impacts on the Hawaiian flora and fauna may occasionally provide neutral (ants) or even beneficial net effects (honeybees), especially in the absence of native birds.     

Germinal center function in the spleen during simian HIV infection in rhesus monkeys

Infection with HIV-1, SIV, or simian HIV is associated with abnormalities in the number, size, and structure of germinal centers (GCs). To determine whether these histopathologic abnormalities are associated with abnormalities in Ab development, we analyzed nucleotide sequences of Igs from splenic GCs of simian HIV-infected macaques. Virus-specific GCs were identified in frozen splenic tissue sections by inverse immunohistochemistry using rHIV-1 gp120 as a probe. B cells from envelope-specific GCs were isolated from these sections using laser capture microdissection. Their Igs were amplified from cDNA using nested PCR, then cloned and sequenced. Nucleotide sequences were recovered from nine multimember clonal lineages. Within each lineage, sequences had similar V-D-J or V-J junctions but differed by somatic mutations distributed throughout the variable domain. The clones were highly mutated, similar to that previously reported for HIV-1-specific human IgG Abs. The average clone had 37 mutations in the V region, for a frequency of 0.11 mutations/base. The mutational pattern was strikingly nonrandom, with somatic mutations occurring preferentially at RGYW/WRCY hotspots. Transition mutations were favored over transversions, with C-->T and G-->A replacements together accounting for almost one-third of all mutations. Analysis of replacement and silent mutations in the framework and CDRs suggests that the Igs were subjected to affinity selection. These data demonstrate that the process of Ab maturation is not seriously disrupted in GCs during the early stages of immunodeficiency virus infection, and that Env-specific Igs developing in GCs are subject to extensive somatic mutation and profound selection pressures.