Site-Specific Bioconjugation of a Murine Dihydrofolate Reductase Enzyme by Copper(I)-Catalyzed Azide-Alkyne Cycloaddition with Retained Activity

Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) is an efficient reaction linking an azido and an alkynyl group in the presence of copper catalyst. Incorporation of a non-natural amino acid (NAA) containing either an azido or an alkynyl group into a protein allows site-specific bioconjugation in mild conditions via CuAAC. Despite its great potential, bioconjugation of an enzyme has been hampered by several issues including low yield, poor solubility of a ligand, and protein structural/functional perturbation by CuAAC components. In the present study, we incorporated an alkyne-bearing NAA into an enzyme, murine dihydrofolate reductase (mDHFR), in high cell density cultivation of Escherichia coli, and performed CuAAC conjugation with fluorescent azide dyes to evaluate enzyme compatibility of various CuAAC conditions comprising combination of commercially available Cu(I)-chelating ligands and reductants. The condensed culture improves the protein yield 19-fold based on the same amount of non-natural amino acid, and the enzyme incubation under the optimized reaction condition did not lead to any activity loss but allowed a fast and high-yield bioconjugation. Using the established conditions, a biotin-azide spacer was efficiently conjugated to mDHFR with retained activity leading to the site-specific immobilization of the biotin-conjugated mDHFR on a streptavidin-coated plate. These results demonstrate that the combination of reactive non-natural amino acid incorporation and the optimized CuAAC can be used to bioconjugate enzymes with retained enzymatic activityope

Controlled Orientation of Active Sites in a Nanostructured Multienzyme Complex

Multistep cascade reactions in nature maximize reaction efficiency by co-assembling related enzymes. Such organization facilitates the processing of intermediates by downstream enzymes. Previously, the studies on multienzyme nanocomplexes assembled on DNA scaffolds demonstrated that closer interenzyme distance enhances the overall reaction efficiency. However, it remains unknown how the active site orientation controlled at nanoscale can have an effect on multienzyme reaction. Here, we show that controlled alignment of active sites promotes the multienzyme reaction efficiency. By genetic incorporation of a non-natural amino acid and two compatible bioorthogonal chemistries, we conjugated mannitol dehydrogenase to formate dehydrogenase with the defined active site arrangement with the residue-level accuracy. The study revealed that the multienzyme complex with the active sites directed towards each other exhibits four-fold higher relative efficiency enhancement in the cascade reaction and produces 60% more D-mannitol than the other complex with active sites directed away from each other.ope

Description of a nationwide structure for monitoring nosocomial outbreaks of (highly resistant) microorganisms in the Netherlands: characteristics of outbreaks in 2012-2021

BACKGROUND: Before 2012, established national surveillance systems in the Netherlands were not able to provide a timely, comprehensive epidemiological view on nosocomial outbreaks. The Healthcare-associated Infections and AntiMicrobial Resistance Monitoring Group (SO-ZI/AMR) was initiated in 2012 for timely national nosocomial outbreak monitoring and risk assessment. This paper aims to describe the achievements of the SO-ZI/AMR by presenting characteristics of outbreaks reported in 2012-2021. METHODS: Hospitals and, since 2015, long-term care facilities (LTCF) were requested to report outbreaks when (1) continuity of care was threatened, or (2) transmission continued despite control measures. A multi-disciplinary expert panel (re-)assessed the public health risk of outbreaks during monthly meetings, using 5 severity phases and based on data collected via standardised questionnaires. We descriptively studied the panel's consensus-based severity classification, distribution of (highly resistant) microorganisms, and duration and size of outbreaks between April 2012 and December 2021. RESULTS: In total, 353 hospital outbreaks and 110 LTCF outbreaks were reported. Most outbreaks (hospitals: n = 309 (88%), LTCF: n = 103 (94%)) did not progress beyond phase 1 (no public health implications, outbreak expected to be controlled within two months), one hospital outbreak reached phase 4 (insufficient/ineffective response: possible public health threat, support offered). Highly resistant microorganisms (HRMO) were involved in 269 (76%) hospital and 103 (94%) LTCF outbreaks. Most outbreaks were caused by methicillin-resistant Staphylococcus aureus (MRSA; n = 93 (26%) in hospitals, n = 80 (72%) in LTCF), vancomycin-resistant Enterococcus faecium (VRE; n = 116 (33%) in hospitals, n = 2 (2%) in LTCF) and highly resistant Enterobacterales (n = 41 (12%) in hospitals, n = 20 (18%) in LTCF). Carbapenemase-producing gram-negative bacteria were involved in 32 (9.1%) hospital and five (4.5%) LTCF outbreaks. In hospitals, VRE outbreaks had the longest duration (median 2.3; range 0.0-22.8 months) and widest range of affected patients (median 9; range 2-483). CONCLUSIONS: The SO-ZI/AMR provided national insight into the characteristics of nosocomial outbreaks over the past decade. HRMO outbreaks - mostly caused by MRSA, VRE (in hospitals) and highly resistant Enterobacterales - occurred regularly, but most of them were controlled quickly and did not develop into a public health threat. The SO-ZI/AMR has become a solid monitoring body, essential to assess risks and raise awareness of potential HRMO threats

Frequency-specific hippocampal-prefrontal interactions during associative learning

Much of our knowledge of the world depends on learning associations (for example, face-name), for which the hippocampus (HPC) and prefrontal cortex (PFC) are critical. HPC-PFC interactions have rarely been studied in monkeys, whose cognitive and mnemonic abilities are akin to those of humans. We found functional differences and frequency-specific interactions between HPC and PFC of monkeys learning object pair associations, an animal model of human explicit memory. PFC spiking activity reflected learning in parallel with behavioral performance, whereas HPC neurons reflected feedback about whether trial-and-error guesses were correct or incorrect. Theta-band HPC-PFC synchrony was stronger after errors, was driven primarily by PFC to HPC directional influences and decreased with learning. In contrast, alpha/beta-band synchrony was stronger after correct trials, was driven more by HPC and increased with learning. Rapid object associative learning may occur in PFC, whereas HPC may guide neocortical plasticity by signaling success or failure via oscillatory synchrony in different frequency bands.National Institute of Mental Health (U.S.) (Conte Center Grant P50-MH094263-03)National Institute of Mental Health (U.S.) (Fellowship F32-MH081507)Picower Foundatio

The “conscious pilot”—dendritic synchrony moves through the brain to mediate consciousness

Cognitive brain functions including sensory processing and control of behavior are understood as “neurocomputation” in axonal–dendritic synaptic networks of “integrate-and-fire” neurons. Cognitive neurocomputation with consciousness is accompanied by 30- to 90-Hz gamma synchrony electroencephalography (EEG), and non-conscious neurocomputation is not. Gamma synchrony EEG derives largely from neuronal groups linked by dendritic–dendritic gap junctions, forming transient syncytia (“dendritic webs”) in input/integration layers oriented sideways to axonal–dendritic neurocomputational flow. As gap junctions open and close, a gamma-synchronized dendritic web can rapidly change topology and move through the brain as a spatiotemporal envelope performing collective integration and volitional choices correlating with consciousness. The “conscious pilot” is a metaphorical description for a mobile gamma-synchronized dendritic web as vehicle for a conscious agent/pilot which experiences and assumes control of otherwise non-conscious auto-pilot neurocomputation

Description of a nationwide structure for monitoring nosocomial outbreaks of (highly resistant) microorganisms in the Netherlands: characteristics of outbreaks in 2012–2021

Abstract Background Before 2012, established national surveillance systems in the Netherlands were not able to provide a timely, comprehensive epidemiological view on nosocomial outbreaks. The Healthcare-associated Infections and AntiMicrobial Resistance Monitoring Group (SO-ZI/AMR) was initiated in 2012 for timely national nosocomial outbreak monitoring and risk assessment. This paper aims to describe the achievements of the SO-ZI/AMR by presenting characteristics of outbreaks reported in 2012–2021. Methods Hospitals and, since 2015, long-term care facilities (LTCF) were requested to report outbreaks when (1) continuity of care was threatened, or (2) transmission continued despite control measures. A multi-disciplinary expert panel (re-)assessed the public health risk of outbreaks during monthly meetings, using 5 severity phases and based on data collected via standardised questionnaires. We descriptively studied the panel’s consensus-based severity classification, distribution of (highly resistant) microorganisms, and duration and size of outbreaks between April 2012 and December 2021. Results In total, 353 hospital outbreaks and 110 LTCF outbreaks were reported. Most outbreaks (hospitals: n = 309 (88%), LTCF: n = 103 (94%)) did not progress beyond phase 1 (no public health implications, outbreak expected to be controlled within two months), one hospital outbreak reached phase 4 (insufficient/ineffective response: possible public health threat, support offered). Highly resistant microorganisms (HRMO) were involved in 269 (76%) hospital and 103 (94%) LTCF outbreaks. Most outbreaks were caused by methicillin-resistant Staphylococcus aureus (MRSA; n = 93 (26%) in hospitals, n = 80 (72%) in LTCF), vancomycin-resistant Enterococcus faecium (VRE; n = 116 (33%) in hospitals, n = 2 (2%) in LTCF) and highly resistant Enterobacterales (n = 41 (12%) in hospitals, n = 20 (18%) in LTCF). Carbapenemase-producing gram-negative bacteria were involved in 32 (9.1%) hospital and five (4.5%) LTCF outbreaks. In hospitals, VRE outbreaks had the longest duration (median 2.3; range 0.0-22.8 months) and widest range of affected patients (median 9; range 2-483). Conclusions The SO-ZI/AMR provided national insight into the characteristics of nosocomial outbreaks over the past decade. HRMO outbreaks – mostly caused by MRSA, VRE (in hospitals) and highly resistant Enterobacterales – occurred regularly, but most of them were controlled quickly and did not develop into a public health threat. The SO-ZI/AMR has become a solid monitoring body, essential to assess risks and raise awareness of potential HRMO threats