51 research outputs found
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One-pot stapling of interchain disulfides of antibodies using an isobutylene motif.
Monoclonal antibodies have emerged as an important class of therapeutics in oncological and autoimmune diseases due to their several attractive properties, such as high binding affinity and specificity. However, it has recently become clear that antibodies recovered from serum show a significantly decreased potency owing to various reasons, including deamidation, oxidation, fragment antigen binding (Fab) exchange, and disulfide shuffling. Fab exchange and disulfide shuffling result because of the instability of disulfides in serum. Herein, we reported a 'one-pot' stapling strategy using isobutylene motifs to stabilise the interchain disulfides of antibodies. This general method was applied to a Fab fragment of the anti-HER2 antibody. The stapled Fab was completely stable in the presence of biological thiols. The approach was further applied to two different full-length IgGs, trastuzumab and rituximab, under mild and biocompatible conditions. The binding affinity of the antibody was enhanced, relative to its native form, after being stapled. The stapled structure maintained its effector functions and behaved similarly to its native form in vivo. This work provides a straightforward and scalable method for the stabilisation of antibodies in various formats
A Biodegradable Polycationic Paint that Kills Bacteria <i>in Vitro</i> and <i>in Vivo</i>
Bacterial
colonization and subsequent formation of biofilms onto surfaces of
medical devices and implants is a major source of nosocomial infections.
Most antibacterial coatings to combat infections are either metal-based
or nondegradable-polymer-based and hence limited by their nondegradability
and unpredictable toxicity. Moreover, to combat infections effectively,
the coatings are required to display simultaneous antibacterial and
antibiofilm activity. Herein we report biocompatible and biodegradable
coatings based on organo-soluble quaternary chitin polymers which
were immobilized noncovalently onto surfaces as bactericidal paint.
The polycationic paint was found to be active against both drug-sensitive
and -resistant bacteria such as methicillin-resistant <i>Staphylococcus
aureus</i> (MRSA), vancomycin-resistant <i>Enterococcus
faecium</i> (VRE), and β-lactam-resistant <i>Klebsiella
pneumoniae</i>. The cationic polymers were shown to interact
with the negatively charged bacterial cell membrane and disrupt the
membrane integrity, thereby causing leakage of intracellular constituents
and cell death upon contact. Importantly, surfaces coated with the
polymers inhibited formation of biofilms against both Gram-positive <i>S. aureus</i> and Gram-negative <i>E. coli</i>, two
of the most clinically important bacteria that form biofilms. Surfaces
coated with the polymers displayed negligible toxicity against human
erythrocytes and embryo kidney cells. Notably, the polymers were shown
to be susceptible toward lysozyme. Furthermore, subcutaneous implantation
of polymer discs in rats led to 15–20% degradation in 4 weeks
thereby displaying their biodegradability. In a murine model of subcutaneous
infection, polymer-coated medical-grade catheter reduced MRSA burden
by 3.7 log compared to that of noncoated catheter. Furthermore, no
biofilm development was observed on the coated catheters under <i>in vivo</i> conditions. The polycationic materials thus developed
herein represent a novel class of safe and effective coating agents
for the prevention of device-associated infections
Gene structure and expression of the aspen cytosolic copper/zinc-superoxide dismutase (PtSodCc1)
Genomic and cDNA clones, corresponding to an ozone-induced cytosolic copper–zinc superoxide dismutase, were isolated from quaking aspen (Populus tremuloides Michx.). The cytosolic superoxide dismutase (SOD) appears to be part of a multi-gene family in aspen and is interrupted by five introns in the coding region. Northern blot analysis with a gene-specific probe revealed an increase in the expression of this gene in response to ozone in the leaves of an ozone-tolerant aspen clone, compared with an ozone-sensitive clone. Cytosolic SOD transcript expression levels in leaves were also found to increase significantly within 6hof mechanical wounding, after which the level of the transcript decreases. Under normal growing conditions, immature male and female aspen floral bud tissues contained the highest levels of the cytosolic SOD gene transcript, whereas transcript levels wer
Cleavable Cationic Antibacterial Amphiphiles: Synthesis, Mechanism of Action, and Cytotoxicities
The development of novel antimicrobial agents having
high selectivity
toward bacterial cells over mammalian cells is urgently required to
curb the widespread emergence of infectious diseases caused by pathogenic
bacteria. Toward this end, we have developed a set of cationic dimeric
amphiphiles (bearing cleavable amide linkages between the headgroup
and the hydrocarbon tail with different methylene spacers) that showed
high antibacterial activity against human pathogenic bacteria (<i>Escherichia coli</i> and <i>Staphylococcus aureus</i>) and low cytotoxicity. The Minimum Inhibitory Concentrations (MIC)
were found to be very low for the dimeric amphiphiles and were lower
or comparable to the monomeric counterpart. In the case of dimeric
amphiphiles, MIC was found to decrease with the increase in the spacer
chain length (<i>n</i> = 2 to 6) and again to increase at
higher spacer length (<i>n</i> > 6). It was found that
the
compound with six methylene spacers was the most active among all
of the amphiphiles (MICs = 10–13 μM). By fluorescence
spectroscopy, fluorescence microscopy, and field-emission scanning
electron microscopy (FESEM), it was revealed that these cationic amphiphiles
interact with the negatively charged bacterial cell membrane and disrupt
the membrane integrity, thus killing the bacteria. All of the cationic
amphiphiles showed low hemolytic activity (HC<sub>50</sub>) and high
selectivity against both gram-positive and gram-negative bacteria.
The most active amphiphile (<i>n</i> = 6) had a 10–13-fold
higher HC<sub>50</sub> than did the MIC. Also, this amphiphile did
not show any cytotoxicity against mammalian cells (HeLa cells) even
at a concentration above the MIC (20 ÎĽM). The critical micellar
concentration (CMC) values of gemini surfactants were found to be
very low (CMC = 0.30–0.11 mM) and were 10–27 times smaller
than the corresponding monomeric analogue (CMC = 2.9 mM). Chemical
hydrolysis and thermogravimetric analysis (TGA) proved that these
amphiphiles are quite stable under both acidic and thermal conditions.
Collectively, these properties make the newly synthesized amphiphiles
potentially superior disinfectants and antiseptics for various biomedical
and biotechnological applications
Small Molecular Antibacterial Peptoid Mimics: The Simpler the Better!
The emergence of multidrug resistant
bacteria compounded by the
depleting arsenal of antibiotics has accelerated efforts toward development
of antibiotics with novel mechanisms of action. In this report, we
present a series of small molecular antibacterial peptoid mimics which
exhibit high in vitro potency against a variety of Gram-positive and
Gram-negative bacteria, including drug-resistant species such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium. The highlight of these compounds
is their superior activity against the major nosocomial pathogen Pseudomonas aeruginosa. Nontoxic toward mammalian
cells, these rapidly bactericidal compounds primarily act by permeabilization
and depolarization of bacterial membrane. Synthetically simple and
selectively antibacterial, these compounds can be developed into a
newer class of therapeutic agents against multidrug resistant bacterial
species
Broad Spectrum Antibacterial and Antifungal Polymeric Paint Materials: Synthesis, Structure–Activity Relationship, and Membrane-Active Mode of Action
Microbial attachment and subsequent
colonization onto surfaces
lead to the spread of deadly community-acquired and hospital-acquired
(nosocomial) infections. Noncovalent immobilization of water insoluble
and organo-soluble cationic polymers onto a surface is a facile approach
to prevent microbial contamination. In the present study, we described
the synthesis of water insoluble and organo-soluble polymeric materials
and demonstrated their structure–activity relationship against
various human pathogenic bacteria including drug-resistant strains
such as methicillin-resistant <i>Staphylococcus aureus</i> (MRSA), vancomycin-resistant enterococci (VRE), and beta lactam-resistant <i>Klebsiella pneumoniae</i> as well as pathogenic fungi such as <i>Candida</i> spp. and <i>Cryptococcus</i> spp. The
polymer coated surfaces completely inactivated both bacteria and fungi
upon contact (5 log reduction with respect to control). Linear polymers
were more active and found to have a higher killing rate than the
branched polymers. The polymer coated surfaces also exhibited significant
activity in various complex mammalian fluids such as serum, plasma,
and blood and showed negligible hemolysis at an amount much higher
than minimum inhibitory amounts (MIAs). These polymers were found
to have excellent compatibility with other medically relevant polymers
(polylactic acid, PLA) and commercial paint. The cationic hydrophobic
polymer coatings disrupted the lipid membrane of both bacteria and
fungi and thus showed a membrane-active mode of action. Further, bacteria
did not develop resistance against these membrane-active polymers
in sharp contrast to conventional antibiotics and lipopeptides, thus
the polymers hold great promise to be used as coating materials for
developing permanent antimicrobial paint
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