Dual-porosity hollow nanoparticles for the immunoprotection and delivery of nonhuman enzymes.

Although enzymes of nonhuman origin have been studied for a variety of therapeutic and diagnostic applications, their use has been limited by the immune responses generated against them. The described dual-porosity hollow nanoparticle platform obviates immune attack on nonhuman enzymes paving the way to in vivo applications including enzyme-prodrug therapies and enzymatic depletion of tumor nutrients. This platform is manufactured with a versatile, scalable, and robust fabrication method. It efficiently encapsulates macromolecular cargos filled through mesopores into a hollow interior, shielding them from antibodies and proteases once the mesopores are sealed with nanoporous material. The nanoporous shell allows small molecule diffusion allowing interaction with the large macromolecular payload in the hollow center. The approach has been validated in vivo using l-asparaginase to achieve l-asparagine depletion in the presence of neutralizing antibodies.

Depletion of perineuronal nets enhances recognition memory and long-term depression in the perirhinal cortex.

Perineuronal nets (PNNs) are extracellular matrix structures surrounding cortical neuronal cell bodies and proximal dendrites and are involved in the control of brain plasticity and the closure of critical periods. Expression of the link protein Crtl1/Hapln1 in neurons has recently been identified as the key event triggering the formation of PNNs. Here we show that the genetic attenuation of PNNs in adult brain Crtl1 knock-out mice enhances long-term object recognition memory and facilitates long-term depression in the perirhinal cortex, a neural correlate of object recognition memory. Identical prolongation of memory follows localized digestion of PNNs with chondroitinase ABC, an enzyme that degrades the chondroitin sulfate proteoglycan components of PNNs. The memory-enhancing effect of chondroitinase ABC treatment attenuated over time, suggesting that the regeneration of PNNs gradually restored control plasticity levels. Our findings indicate that PNNs regulate both memory and experience-driven synaptic plasticity in adulthood.

Prevalence and molecular epidemiological typing of penicillinase-producing Neisseria gonorrhoeae and their bla(TEM-135) gene variants in Nanjing, China.

BACKGROUND: This study aimed to investigate the prevalence of penicillinase-producing Neisseria gonorrhoeae (PPNG) and their blaTEM-135 gene variant in 2007 and 2012 in Nanjing, China. In addition, molecular epidemiological typing of all isolates was performed to elucidate the genetic relationships of the PPNG strains. METHODS: A total of 199 and 77 N. gonorrhoeae isolates were collected at the National Center for STD Control in 2007 and 2012, respectively. Nitrocefin tests were performed to identify PPNG. Mismatch amplification mutation assay was used to identify blaTEM-135. All isolates were genotyped using N. gonorrhoeae multiantigen sequence typing (NG-MAST), and additionally, porB-based phylogenetic analysis was performed for the PPNG isolates. RESULTS: The total prevalence of PPNG isolates was 41% (114/276) and 58% (66/114) of these PPNG isolates possessed bla(TEM-135). In 2007, 45% (90/199) produced ß-lactamase, and of those PPNG, 58% (52/90) possessed bla(TEM-135). In 2012, 31% (24/77) were PPNG, and 58% (14/24) of those isolates contained bla(TEM-135). There were 162 NG-MAST STs among the 276 isolates, and 89 of those were novel STs. A strong association between specific NG-MAST STs and bla(TEM-135) was found, and the porB-based phylogenetic analysis showed a distant evolutionary relationship between isolates in 2007 and isolates in 2012. CONCLUSIONS: A high prevalence of PPNG and blaTEM-135 was found in Nanjing, China. bla(TEM-135) might be a precursor in the evolution into an extended-spectrum ß-lactamase that can degrade ceftriaxone, which stresses the need to continuously monitor PPNG, blaTEM-135, and additional evolving blaTEM gene variants.

Impaired Cognitive Function after Perineuronal Net Degradation in the Medial Prefrontal Cortex.

Perineuronal nets (PNNs) are highly organized components of the extracellular matrix that surround a subset of mature neurons in the CNS. These structures play a critical role in regulating neuronal plasticity, particularly during neurodevelopment. Consistent with this role, their presence is associated with functional and structural stability of the neurons they ensheath. A loss of PNNs in the prefrontal cortex (PFC) has been suggested to contribute to cognitive impairment in disorders such as schizophrenia. However, the direct consequences of PNN loss in medial PFC (mPFC) on cognition has not been demonstrated. Here, we examined behavior after disruption of PNNs in mPFC of Long-Evans rats following injection of the enzyme chondroitinase ABC (ChABC). Our data show that ChABC-treated animals were impaired on tests of object oddity perception. Performance in the cross-modal object recognition (CMOR) task was not significantly different for ChABC-treated rats, although ChABC-treated rats were not able to perform above chance levels whereas control rats were. ChABC-treated animals were not significantly different from controls on tests of prepulse inhibition (PPI), set-shifting (SS), reversal learning, or tactile and visual object recognition memory. Posthumous immunohistochemistry confirmed significantly reduced PNNs in mPFC due to ChABC treatment. Moreover, PNN density in the mPFC predicted performance on the oddity task, where higher PNN density was associated with better performance. These findings suggest that PNN loss within the mPFC impairs some aspects of object oddity perception and recognition and that PNNs contribute to cognitive function in young adulthood.

Perineuronal Nets Enhance the Excitability of Fast-Spiking Neurons.

Perineuronal nets (PNNs) are specialized complexes of extracellular matrix molecules that surround the somata of fast-spiking neurons throughout the vertebrate brain. PNNs are particularly prevalent throughout the auditory brainstem, which transmits signals with high speed and precision. It is unknown whether PNNs contribute to the fast-spiking ability of the neurons they surround. Whole-cell recordings were made from medial nucleus of the trapezoid body (MNTB) principal neurons in acute brain slices from postnatal day 21 (P21) to P27 mice. PNNs were degraded by incubating slices in chondroitinase ABC (ChABC) and were compared to slices that were treated with a control enzyme (penicillinase). ChABC treatment did not affect the ability of MNTB neurons to fire at up to 1000 Hz when driven by current pulses. However, f-I (frequency-intensity) curves constructed by injecting Gaussian white noise currents superimposed on DC current steps showed that ChABC treatment reduced the gain of spike output. An increase in spike threshold may have contributed to this effect, which is consistent with the observation that spikes in ChABC-treated cells were delayed relative to control-treated cells. In addition, parvalbumin-expressing fast-spiking cortical neurons in >P70 slices that were treated with ChABC also had reduced excitability and gain. The development of PNNs around somata of fast-spiking neurons may be essential for fast and precise sensory transmission and synaptic inhibition in the brain.

Effect of clavulanic acid on inactivated amoxicillin excretion in urinary tract infection.

Oral amoxicillin was taken with and without clavulanic acid by normal subjects and by patients with chronic complicated urinary tract infection to examine the in vitro protective effect of clavulanic acid on amoxicillin degradation. When amoxicillin alone was taken, urinary excretion of the penicilloic acid of amoxicillin in bacteria-positive patients was higher than that in bacteria-negative patients and in normal subjects. There was no comparable change in urinary penicilloic acid excretion in the presence of clavulanic acid. There were significant in vitro protective effects of clavulanic acid on beta-lactamases in the urine.

Evaluation of Stuart's transport medium containing penicillinase. An in vitro study. Brief report.

An in vitro study was performed to evaluate the value of penicillinase in Stuart's transport medium. Swabs were taken from serum broth cultures of Staphylococcus aureus, Escherichia coli and Proteus rettgeri, incubated with cefuroxime. Following 24 hours' storage in the transport medium, the recovery of P. rettgeri was significantly higher from Stuart's transport media containing penicillinase in a concentration of 100,000 units/ml, than from swabs stored in Stuart's medium without penicillinase. Although susceptible to cefuroxime, we did not find the same effect on S. aureus and E. coli, presumably because these strains had higher MIC values than the P. rettgeri strain tested.

Effect of antibiotic resistance on the survival of Staphylococcus aureus.

The survival of strains of Staphylococcus aureus on glass at 30 degrees C, 37 degrees C, and room temperature was compared with derivatives of them that had either lost or gained naturally occurring antibiotic resistance. In other properties the sets of strains were identical. Neither loss nor gain of antibiotic resistance (methicillin, penicillinase, streptomycin, tetracycline, erythromycin, neomycin) altered survival.

Progress towards agreement in reports of antibiotic sensitivity.

Results of three quality control trials of antibiotic sensitivity testing carried out on staphylococci by the Birmingham Regional Bacteriologists Group are presented. An overall reduction in the number of discrepant results from 5.2 to 1.2% when retesting the same organisms was thought to be due largely to the alteration in methods of testing for resistance to neomycin- and penicillinase-resistant antibiotics. Improvements were noted in the performance of all laboratories, which were felt to be due partly to participation in the trials.

Further evolution of a strain of Staphylococcus aureus in vivo: evidence for significant inactivation of flucloxacillin by penicillinase.

A strain of Staphylococcus aureus (no. FAR4) has been isolated at intervals, for 32 months, from the sputum of a patient with cystic fibrosis of the lung. Changes in the properties of isolates of this strain over the first 18 months have been reported previously (Lacey et al., 1973 and 1974). During the last 14 months (May 1973 to July 1974), further evolution has occurred to produce a total of 31 distinct phenotypes. Recent changes are as follows. 1. The ability of isolates to produce penicillinase in vitro was closely correlated with flucloxacillin therapy. Inactivation of flucloxacillin by penicillinase was demonstrated by diffusion testing (but not MIC determination) in vitro and may have occurred to a significant extent in vivo. 2. Lincomycin-resistant mutants slowly disappeared from the sputum after the termination of clindamycin therapy. 3. All of the recent isolates were resistant to erythromycin, possibly because of the linkage of the genes coding for erythromycin resistance with those coding for the production of delta-haemolysin; delta-haemolysin may be an important "virulence factor".

Naturally-occurring, osmo-remedial variants of Escherichia coli.

Two clones of Escherichia coli O27:K1:H31 and O2:H7, isolated from patients with urinary tract infection or bacteraemia, failed to grow in a synthetic minimal medium (MM) of low osmolality. They were considered to be osmo-remedial because they grew well when sufficient amounts of NaCl, mannitol or sucrose were added to raise the osmolality of the medium to > 300 mOsm/kg. The defect could also be corrected by nicotinamide or its precursors quinolinic and aspartic acids. Each clone had a unique DNA restriction enzyme profile, fimbriae and antibiotic susceptibility patterns. The osmo-remedial variants were unstable and underwent phenotypic modulation to form mixtures with osmo-tolerant forms when grown in MM. They tended to form satellites of small colonies around large colonies of osmo-tolerant cells on MM agar plates. The penicillin method of Davis was used to separate the two forms. Nicotinamide induced the expression of ompF when the osmo-remedial strains were grown under conditions of low osmolality. It is possible that the variants are defective in the synthesis of membrane-derived oligosaccharides or outer-membrane proteins, but this has yet to be determined.

Effect of penicillin on an excitatory synapse.

When penicillin, an epileptogenic agent, was applied to the neuromuscular junctions of the superficial flexor muscles of crayfish, the excitatory junctional potential (EJP) amplitudes were increased by 50-200%. This effect of the drug was not due to changes in the passive electrical properties of the muscle cell membrane or to an increase in its chemical sensitivity to acetylcholine (ACh), the presumed transmitter at the junction studied. Inactivating the penicillin with the enzyme penicillinase, or substituting acetate for penicillin in the test solutions eliminated the effect on EJPs, showing that the penicillin ion was the active agent. Penicillin ions did decrease the frequency of spontaneous miniature EJPs and increase the amplitude or presynaptic spikes recorded extracellularly, suggesting that augmentation of EJPs may have been due to alterations at the presynaptic nerve terminals.

Controversies in antimicrobial susceptibility testing.

This article examines a number of areas of disagreement surrounding antimicrobial susceptibility testing and discusses some of the useful susceptibility testing techniques for which no standardized procedures have been established.

Enzymatic conversion of the unnatural tripeptide delta-(D-alpha-aminoadipyl)-L-cysteinyl-D-valine to beta-lactam antibiotics.

Incubation of the unnatural tripeptide delta-(D-alpha-aminoadipyl)-L-cysteinyl-D-valine (DLD-ACV) with a partially purified extract of Cephalosporium acremonium resulted in the production of deacetoxycephalosporin C. The extract contained isopenicillin N synthetase (cyclase) and deacetoxycephalosporin C synthetase (expandase) but no penicillin epimerase activity, and was incubated aerobically in the presence of the components of the cyclase and expandase reaction mixtures (Fe++, ascorbate, dithiothreitol, alpha-ketoglutarate and ATP). The reaction was sensitive to penicillinase, indicating penicillin N to be an intermediate. However, when ring expansion was prevented by omission of alpha-ketoglutarate and ATP, no penicillin N was detected.

[Current microbiological problems. Antibiotic resistance and therapeutic problems raised by Pseudomonas aeruginosa]. / Problèmes microbiologiques d'actualité. Résistance aux antibiotiques et problèmes thérapeutiques posés par Pseudomonas aeruginosa.

RESISTANCE: Pseudomonas aeruginosa is characterized by its low intrinsic susceptibility to many antibiotics and its capacity to acquire additional resistance mechanisms to usually active drugs. Some beta-lactam resistance mechanisms are well known (penicillinase production, cephalosporinase overproduction) and others have been recently identified, such as active efflux systems, which confer coresistance to quinolones, and new beta-lactamases which are limited to a few countries (extended-spectrum beta-lactamases, imipenemase). Ceftazidime remains the most active beta-lactam agent. ACTIVE DRUGS: Among aminoglycosides, amikacin and isepamicin are the most frequently active drugs. The use of fluoroquinolones is limited by a high incidence of acquired resistance. The percentage of resistant strains is highly variable according to countries, hospitals and wards. CLINICAL PRACTICE: Therapy, usually based on a beta-lactam-aminoglycoside combination, will be empirical at first, according to local epidemiological factors, site of infection and previously administered antibiotics, then re-evaluated according to susceptibility results.