Regulatory Considerations in the Approval of Rezafungin (Rezzayo) for the Treatment of Candidemia and Invasive Candidiasis in Adults.

On 22 March 2023, the FDA approved rezafungin (Rezzayo) for the treatment of candidemia and invasive candidiasis in adults with limited or no alternative treatment options. Rezafungin is an echinocandin that supports weekly dosing, enabling outpatient parenteral treatment that potentially avoids the need for a central venous catheter. Approval of rezafungin was based on a single adequate and well-controlled phase 3 study designed with a day 30 all-cause mortality primary end point and 20% noninferiority margin, which demonstrated that rezafungin is noninferior to the comparator echinocandin. Nonclinical studies of rezafungin in nonhuman primates identified a neurotoxicity safety signal; however, rezafungin's safety profile in the completed clinical studies was similar to other Food and Drug Administration-approved echinocandins. Here we describe the rationale for this approval and important considerations during the review process for a flexible development program intended to expedite the availability of antimicrobial therapies to treat serious infections in patients with limited treatment options. Clinical Trials Registration . NCT02734862 and NCT03667690.

Microfabrication of a Chamber for High-Resolution, In Situ Imaging of the Whole Root for Plant-Microbe Interactions.

Fabricated ecosystems (EcoFABs) offer an innovative approach to in situ examination of microbial establishment patterns around plant roots using nondestructive, high-resolution microscopy. Previously high-resolution imaging was challenging because the roots were not constrained to a fixed distance from the objective. Here, we describe a new 'Imaging EcoFAB' and the use of this device to image the entire root system of growing Brachypodium distachyon at high resolutions (20×, 40×) over a 3-week period. The device is capable of investigating root-microbe interactions of multimember communities. We examined nine strains of Pseudomonas simiae with different fluorescent constructs to B. distachyon and individual cells on root hairs were visible. Succession in the rhizosphere using two different strains of P. simiae was examined, where the second addition was shown to be able to establish in the root tissue. The device was suitable for imaging with different solid media at high magnification, allowing for the imaging of fungal establishment in the rhizosphere. Overall, the Imaging EcoFAB could improve our ability to investigate the spatiotemporal dynamics of the rhizosphere, including studies of fluorescently-tagged, multimember, synthetic communities.

Conservation and Diversity in the Primary Reverse Photodynamics of the Canonical Red/Green Cyanobacteriochrome Family.

In this report, we compare the femtosecond to nanosecond primary reverse photodynamics (15EPg → 15ZPr) of eight tetrapyrrole binding photoswitching cyanobacteriochromes in the canonical red/green family from the cyanobacterium Nostoc punctiforme. Three characteristic classes were identified on the basis of the diversity of excited-state and ground-state properties, including the lifetime, photocycle initiation quantum yield, photointermediate stability, spectra, and temporal properties. We observed a correlation between the excited-state lifetime and peak wavelength of the electronic absorption spectrum with higher-energy-absorbing representatives exhibiting both faster excited-state decay times and higher photoisomerization quantum yields. The latter was attributed to both an increased number of structural restraints and differences in H-bonding networks that facilitate photoisomerization. All three classes exhibited primary Lumi-Go intermediates, with class II and III representatives evolving to a secondary Meta-G photointermediate. Class II Meta-GR intermediates were orange absorbing, whereas class III Meta-G had structurally relaxed, red-absorbing chromophores that resemble their dark-adapted 15ZPr states. Differences in the reverse and forward reaction mechanisms are discussed within the context of structural constraints.

Forward Photodynamics of the Noncanonical Red/Green NpR3784 Cyanobacteriochrome from Nostoc punctiforme.

Cyanobacteriochromes (CBCRs) make up a diverse family of cyanobacterial photoreceptors distantly related to the phytochrome photoreceptors of land plants. At least two lineages of CBCRs have reacquired red-absorbing dark states similar to the phytochrome Pr resting state but are coupled to green-absorbing light-adapted states rather than the canonical far-red-absorbing light-adapted state. One such lineage includes the canonical red/green (R/G) CBCRs that includes AnPixJg2 (UniProtKB Q8YXY7 ) and NpR6012g4 (UniProtKB B2IU14 ) that have been extensively characterized. Here we examine the forward Pr photodynamics of NpR3784 (UniProtKB B2J457 ), a representative member of the second R/G CBCR subfamily. Using broadband transient absorption pump-probe spectroscopy, we characterize both primary (100 fs to 10 ns) and secondary (10 ns to 1 ms) forward (Pr → Pg) photodynamics and compare the results to temperature-jump cryokinetics measurements. Our studies show that primary isomerization dynamics occur on an ∼10 ps timescale, yet remarkably, the red-shifted primary Lumi-Rf photoproduct found in all photoactive canonical R/G CBCRs examined to date is extremely short-lived in NpR3784. These results demonstrate that differences in reaction pathways reflect the evolutionary history of R/G CBCRs despite the convergent evolution of their photocycle end products.

Reverse Photodynamics of the Noncanonical Red/Green NpR3784 Cyanobacteriochrome from Nostoc punctiforme.

In the companion paper (10.1021/acs.biochem.8b01274), we examined the forward Pr photodynamics of NpR3784 (UniProtKB B2J457 ), a representative member of a noncanonical red/green (R/G) cyanobacteriochrome (CBCR) subfamily. Here the reverse Pg → Pr photodynamics of NpR3784 was studied by broadband transient absorption pump-probe spectroscopy. Primary (100 fs to 10 ns) and secondary (10 ns to 1 ms) photodynamics were characterized over nine decades of time, which also were complemented with temperature-jump cryokinetics measurements. In contrast with canonical R/G CBCRs, the NpR3784 reverse photoconversion yielded two spectrally distinct primary photoproducts, Lumi-Go and Lumi-Gr, which decay on different time scales. The two primary photoproducts of NpR3784 equilibrate on the 40 ns time scale and subsequently propagate as a single intermediate population into Pr. Such heterogeneity could arise from differences in the direction of D-ring rotation, in chromophore protonation or hydrogen bonding, or in the mobility of protein residues or of solvent water nearby the chromophore or some combination therein. We conclude that the atypical photodynamics of NpR3784 reflects chromophore-protein interactions that differ from those present in the canonical R/G CBCR family.

Conservation and diversity in the primary forward photodynamics of red/green cyanobacteriochromes.

Phytochromes are red/far-red photosensory proteins that detect the ratio of red to far-red light. Crucial to light regulation of plant developmental biology, phytochromes are also found in fungi, bacteria, and eukaryotic algae. In addition to phytochromes, cyanobacteria also can contain distantly related cyanobacteriochromes (CBCRs) that, like phytochromes, utilize the photoisomerization of a linear tetrapyrrole (bilin) chromophore to convert between two photostates with distinct spectral properties. CBCRs exhibit a wide range of photostates spanning the visible and even near-ultraviolet spectrum. In both phytochromes and CBCRs, biosynthesis initially yields a holoprotein with bilin in the 15Z configuration, and the 15E photoproduct can often revert to the 15Z photostate in the absence of light (dark reversion). One CBCR subfamily, red/green CBCRs, typically exhibits red-absorbing dark states and green-absorbing photoproducts. Dark reversion is extremely variable in red/green CBCRs with known examples ranging from seconds to days. One red/green CBCR, NpR6012g4 from Nostoc punctiforme, is also known to exhibit forward photoconversion that has an unusually high quantum yield at ∼40% compared to 10-20% for phytochromes and CBCRs from other subfamilies. In the current study, we use time-resolved pump-probe absorption spectroscopy with broadband detection and multicomponent global analysis to characterize forward photoconversion of seven additional red/green CBCRs from N. punctiforme on an ultrafast time scale. Our results reveal that red/green CBCRs exhibit a conserved pathway for primary forward photoconversion but that considerable diversity exists in their excited-state lifetimes, photochemical quantum yields, and primary photoproduct stabilities.

Dynamic inhomogeneity in the photodynamics of cyanobacterial phytochrome Cph1.

Phytochromes are widespread red/far-red photosensory proteins well known as critical regulators of photomorphogenesis in plants. It is often assumed that natural selection would have optimized the light sensing efficiency of phytochromes to minimize nonproductive photochemical deexcitation pathways. Surprisingly, the quantum efficiency for the forward Pr-to-Pfr photoconversion of phytochromes seldom exceeds 15%, a value very much lower than that of animal rhodopsins. Exploiting ultrafast excitation wavelength- and temperature-dependent transient absorption spectroscopy, we resolve multiple pathways within the ultrafast photodynamics of the N-terminal PAS-GAF-PHY photosensory core module of cyanobacterial phytochrome Cph1 (termed Cph1Δ) that are primarily responsible for the overall low quantum efficiency. This inhomogeneity primarily reflects a long-lived fluorescent subpopulation that exists in equilibrium with a spectrally distinct, photoactive subpopulation. The fluorescent subpopulation is favored at elevated temperatures, resulting in anomalous excited-state dynamics (slower kinetics at higher temperatures). The spectral and kinetic behavior of the fluorescent subpopulation strongly resembles that of the photochemically compromised and highly fluorescent Y176H variant of Cph1Δ. We present an integrated, heterogeneous model for Cph1Δ that is based on the observed transient and static spectroscopic signals. Understanding the molecular basis for this dynamic inhomogeneity holds potential for rational design of efficient phytochrome-based fluorescent and photoswitchable probes.

Heterogeneous photodynamics of the pfr state in the cyanobacterial phytochrome Cph1.

Femtosecond photodynamics of the Pfr form of the red/far-red phytochrome N-terminal PAS-GAF-PHY photosensory core module of the cyanobacterial phytochrome Cph1 (termed Cph1Δ) from Synechocystis were resolved with visible broadband transient absorption spectroscopy. Multiphasic generation dynamics via global target analysis revealed parallel evolution of two pathways with distinct excited- and ground-state kinetics. These measurements resolved two subpopulations: a majority subpopulation with fast excited-state decay and slower ground-state dynamics, corresponding to previous descriptions of Pfr dynamics, and a minority subpopulation with slower excited-state decay and faster ground-state primary dynamics. Both excited-state subpopulations generated the isomerized, red-shifted Lumi-Ff photoproduct (715 nm); subsequent ground-state evolution to a blue-shifted Meta-Fr population (635 nm) proceeded on 3 ps and 1.5 ns time scales for the two subpopulations. Meta-Fr was spectrally similar to a recently described photoinactive fluorescent subpopulation of Pr ((Fluor)Pr). Thus, the reverse Pfr to Pr photoconversion of Cph1Δ involves minor structural deformation of Meta-Fr to generate the fluorescent, photochemically refractory form of Pr, with slower subsequent equilibration with the photoactive Pr subpopulation ((Photo)Pr).

Primary and secondary photodynamics of the violet/orange dual-cysteine NpF2164g3 cyanobacteriochrome domain from Nostoc punctiforme.

Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors distantly related to phytochromes. Like phytochromes, CBCRs photointerconvert between two photostates that accompany photoisomerization of their bilin chromophores. While phytochromes typically exhibit red/far-red photocycles, CBCR photocycles are much more diverse, spanning the near-ultraviolet and the entire visible region. All CBCRs described to date have a conserved Cys residue covalently attached to the linear tetrapyrrole (bilin) chromophore; two CBCR subfamilies also exploit a second thioether linkage to the chromophore for detection of near-ultraviolet to blue light. Here, we present the photodynamic analysis of the insert-Cys CBCR NpF2164g3, a representative of the second class of two-cysteine CBCRs. Using broadband transient absorption pump-probe spectroscopy, we characterize the primary (100 fs to 10 ns) and secondary (10 ns to 1 ms) photodynamics in both directions, examining photodynamics over nine decades of time. Primary isomerization dynamics occur on a ~10 ps time scale for both forward and reverse reactions. In contrast to previous studies on Tlr0924, a representative of the other class of two-cysteine CBCRs, formation and elimination of the second linkage are slower than the 1 ms experimental range probed here. These results extend our understanding of dual-cysteine CBCR photocycles in the phytochrome superfamily.

Primary photodynamics of the green/red-absorbing photoswitching regulator of the chromatic adaptation E domain from Fremyella diplosiphon.

Phytochromes are red/far-red photosensory proteins that utilize the photoisomerization of a linear tetrapyrrole (bilin) chromophore to detect the red to far-red light ratio. Cyanobacteriochromes (CBCRs) are distantly related cyanobacterial photosensors with homologous bilin-binding GAF domains, but they exhibit greater spectral diversity. Different CBCR subfamilies have been described, with spectral sensitivity varying across the near-ultraviolet and throughout the visible spectrum, but all known CBCRs utilize photoisomerization of the bilin 15,16-double bond as the primary photochemical event. The first CBCR discovered was RcaE, responsible for tuning light harvesting to the incident color environment (complementary chromatic adaptation) in Fremyella diplosiphon. The green/red RcaE photocycle has recently been described in detail. We now extend this analysis by examining femtosecond photodynamics using ultrafast transient absorption techniques with broadband detection and multicomponent global analysis. Excited-state dynamics in both directions are significantly slower than those recently published for the red/green CBCR NpR6012g4. In the forward reaction, the primary Lumi-G photoproduct arises from the longer-lived excited-state populations, leading to a low photoproduct quantum yield. Using dual-excitation wavelength interleaved pump-probe spectroscopy, we observe multiphasic excited-state dynamics in the forward reaction ((15Z)Pg → (15E)Pr), which we interpret as arising from ground-state inhomogeneity with different tautomers of the PCB chromophore. The reverse reaction ((15E)Pr → (15Z)Pg) is characterized via pump-probe spectroscopy and also exhibits slow excited-state decay dynamics and a low photoproduct yield. These results provide the first description of excited-state dynamics for a green/red CBCR.

Mutations in proteasome subunit ß type 8 cause chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature with evidence of genetic and phenotypic heterogeneity.

OBJECTIVE: Chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE syndrome) is an autoinflammatory syndrome recently described in children. We undertook this study to investigate the clinical phenotype, genetic cause, and immune dysregulation in 9 CANDLE syndrome patients. METHODS: Genomic DNA from all patients was screened for mutations in PSMB8 (proteasome subunit ß type 8). Cytokine levels were measured in sera from 3 patients. Skin biopsy samples were evaluated by immunohistochemistry, and blood microarray profile and STAT-1 phosphorylation were assessed in 4 patients and 3 patients, respectively. RESULTS: One patient was homozygous for a novel nonsense mutation in PSMB8 (c.405C>A), suggesting a protein truncation; 4 patients were homozygous and 2 were heterozygous for a previously reported missense mutation (c.224C>T); and 1 patient showed no mutation. None of these sequence changes was observed in chromosomes from 750 healthy controls. Of the 4 patients with the same mutation, only 2 shared the same haplotype, indicating a mutational hot spot. PSMB8 mutation-positive and -negative patients expressed high levels of interferon-γ (IFNγ)-inducible protein 10. Levels of monocyte chemotactic protein 1, interleukin-6 (IL-6), and IL-1 receptor antagonist were moderately elevated. Microarray profiles and monocyte STAT-1 activation suggested a unique IFN signaling signature, unlike in other autoinflammatory disorders. CONCLUSION: CANDLE syndrome is caused by mutations in PSMB8, a gene recently reported to cause "JMP" syndrome (joint contractures, muscle atrophy, microcytic anemia, and panniculitis-induced childhood-onset lipodystrophy) in adults. We extend the clinical and pathogenic description of this novel autoinflammatory syndrome, thereby expanding the clinical and genetic disease spectrum of PSMB8-associated disorders. IFN may be a key mediator of the inflammatory response and may present a therapeutic target.

A combinatorial droplet microfluidic device integrated with mass spectrometry for enzyme screening.

Mass spectrometry (MS) enables detection of different chemical species with a very high specificity; however, it can be limited by its throughput. Integrating MS with microfluidics has a tremendous potential to improve throughput and accelerate biochemical research. In this work, we introduce Drop-NIMS, a combination of a passive droplet loading microfluidic device and a matrix-free MS laser desorption ionization technique called nanostructure-initiator mass spectrometry (NIMS). This platform combines different droplets at random to generate a combinatorial library of enzymatic reactions that are deposited directly on the NIMS surface without requiring additional sample handling. The enzyme reaction products are then detected with MS. Drop-NIMS was used to rapidly screen enzymatic reactions containing low (on the order of nL) volumes of glycoside reactants and glycoside hydrolase enzymes per reaction. MS "barcodes" (small compounds with unique masses) were added to the droplets to identify different combinations of substrates and enzymes created by the device. We assigned xylanase activities to several putative glycoside hydrolases, making them relevant to food and biofuel industrial applications. Overall, Drop-NIMS is simple to fabricate, assemble, and operate and it has potential to be used with many other small molecule metabolites.

Femtosecond photodynamics of the red/green cyanobacteriochrome NpR6012g4 from Nostoc punctiforme. 1. Forward dynamics.

Phytochromes are well-known red/far-red photosensory proteins that utilize the photoisomerization of a linear tetrapyrrole (bilin) chromophore to detect the ratio of red to far-red light. Cyanobacteriochromes (CBCRs) are related photosensory proteins with a bilin-binding GAF domain, but much more diverse spectral sensitivity, with five recognized subfamilies of CBCRs described to date. The mechanisms that underlie this spectral diversity have not yet been fully elucidated. One of the main CBCR subfamilies photoconverts between a red-absorbing ground state, like the familiar P(r) state of phytochromes, and a green-absorbing photoproduct (P(g)). Here, we examine the ultrafast forward photodynamics of the red/green CBCR NpR6012g4 from the NpR6012 locus of the nitrogen-fixing cyanobacterium Nostoc punctiforme. Using transient absorption spectroscopy with broadband detection and multicomponent global analysis, we observed multiphasic excited-state dynamics that induces the forward reaction (red-absorbing to green-absorbing), which we interpret as arising from ground-state heterogeneity. Excited-state decays with lifetimes of 55 and 345 ps generate the primary photoproduct (Lumi-R), and the fastest decay (5 ps) did not produce Lumi-R. Although the photoinduced kinetics of Npr6012g4 is comparable with that of the Cph1 phytochrome isolated from Synechocystis cyanobacteria, NpR6012g4 exhibits a ≥2-3-fold higher photochemical quantum yield. Understanding the structural basis of this enhanced quantum yield may prove to be useful in increasing the photochemical efficiency of other bilin-based photosensors.

Femtosecond photodynamics of the red/green cyanobacteriochrome NpR6012g4 from Nostoc punctiforme. 2. reverse dynamics.

Phytochromes are red/far-red photosensory proteins that utilize photoisomerization of a linear tetrapyrrole (bilin) chromophore to photoconvert reversibly between red- and far-red-absorbing forms (P(r) and P(fr), respectively). Cyanobacteriochromes (CBCRs) are related photosensory proteins with more diverse spectral sensitivity. The mechanisms that underlie this spectral diversity have not yet been fully elucidated. One of the main CBCR subfamilies photoconverts between a red-absorbing 15Z ground state, like the familiar P(r) state of phytochromes, and a green-absorbing photoproduct ((15E)P(g)). We have previously used the red/green CBCR NpR6012g4 from the cyanobacterium Nostoc punctiforme to examine ultrafast photodynamics of the forward photoreaction. Here, we examine the reverse reaction. Using excitation-interleaved transient absorption spectroscopy with broadband detection and multicomponent global analysis, we observed multiphasic excited-state dynamics. Interleaved excitation allowed us to identify wavelength-dependent shifts in the ground-state bleach that equilibrated on a 200 ps time scale, indicating ground-state heterogeneity. Compared to the previously studied forward reaction, the reverse reaction has much faster excited-state decay time constants and significantly higher photoproduct yield. This work thus demonstrates striking differences between the forward and reverse reactions of NpR6012g4 and provides clear evidence of ground-state heterogeneity in the phytochrome superfamily.

Second-chance forward isomerization dynamics of the red/green cyanobacteriochrome NpR6012g4 from Nostoc punctiforme.

The primary ultrafast Z-to-E isomerization photodynamics of the phytochrome-related cyanobacteriochrome NpR6012g4 from Nostoc punctiforme was studied by transient absorption pump-dump-probe spectroscopy. A 2 ps dump pulse resonant with the stimulated emission band depleted 21% of the excited-state population, while the initial photoproduct Lumi-R was depleted by only 11%. We observed a red-shifted ground-state intermediate (GSI) that we assign to a metastable state that failed to isomerize fully. Multicomponent global analysis implicates the generation of additional Lumi-R from the GSI via crossing over the ground-state thermal barrier for full isomerization, explaining the discrepancy between excited-state and Lumi-R depletion by the dump pulse. This second-chance ground-state dynamics provides a plausible explanation for the unusually high quantum yield of 40% for the primary isomerization step in the forward reaction of NpR6012g4.

A novel mutation in the interleukin-1 receptor antagonist associated with intrauterine disease onset.

Deficiency of the IL-1 receptor antagonist (DIRA) is a recently described rare autoinflammatory disease, caused by loss of function mutations in IL1RN leading to the unopposed activation of the IL-1 pathway. We describe a novel nonsense mutation in the IL1RN gene, associated with early intrauterine onset, death and multiorgan involvement in a prematurely born baby. The protein prediction model indicated that the novel Q119X mutation would result in a nonfunctional protein by impairing the ability of the IL-1Ra to bind and antagonize signaling through the IL-1R. Since the disorder may mimic severe bacterial infections and the treatment with anakinra is life saving, we intend to raise awareness of the syndrome and the possibility of a founder mutation that may lead to the diagnosis of additional cases in Turkey. The clinical suspicion of DIRA is critical to avoid improper management of the patients with antibiotics alone and death from multiorgan failure.

A novel mutation of IL1RN in the deficiency of interleukin-1 receptor antagonist syndrome: description of two unrelated cases from Brazil.

OBJECTIVE: Monogenic autoinflammatory diseases are disorders of Mendelian inheritance that are characterized by mutations in genes that regulate innate immunity and whose typical features are systemic inflammation without high-titer autoantibodies or antigen-specific T cells. Skin and bone inflammation in the newborn period have been described in 3 of these autoinflammatory disorders: neonatal-onset multisystem inflammatory disease, Majeed syndrome, and deficiency of interleukin-1 (IL-1) receptor antagonist (DIRA) syndrome. This study was undertaken to present the characteristics of the DIRA syndrome in 2 cases from Brazil, and describe a novel mutation in IL1RN. METHODS: Two unrelated Brazilian patients were evaluated for the clinical signs and symptoms of these 3 disorders, and peripheral blood samples were assessed for mutations in NLRP3, LPIN2, and IL1RN by DNA resequencing analysis. A mutation in IL1RN that encodes a mutant protein was identified, and the expression and function of this mutant protein were compared to those of the wild-type protein. RESULTS: Both patients presented with pustular dermatitis resembling generalized pustular psoriasis, recurrent multifocal aseptic osteomyelitis, and elevation in the levels of acute-phase reactants, all of which are features most consistent with the DIRA syndrome. Chronic lung disease was observed in 1 of the patients, and jugular venous thrombosis was observed in the other patient. Both patients showed a partial response to corticosteroid therapy, and 1 patient experienced an initial improvement of dermatitis with the use of acitretin. Both patients were homozygous for a novel 15-bp (in-frame) deletion on the IL1RN gene. The mutated protein expressed in vitro had no affinity with the IL-1 receptor, and stimulation of the patients' cells with recombinant human IL-1α or IL-1ß led to oversecretion of proinflammatory cytokines, similar to the findings obtained in previously reported patients. CONCLUSION: The presence of the same homozygous novel mutation in IL1RN in 2 unrelated Brazilian patients suggests that this genetic variant may be a founder mutation that has been introduced in the Brazilian population.

Ultrafast E to Z photoisomerization dynamics of the Cph1 phytochrome.

Femtosecond photodynamics of the reverse ( 15E Pfr→ 15Z Pr) reaction of the red/far-red phytochrome Cph1 from Synechocystis were resolved with visible broadband transient absorption spectroscopy. Multi-phasic dynamics were resolved and separated via global target analysis into a fast-decaying (260 fs) excited-state population that bifurcates to generate the isomerized Lumi-F primary photoproduct and a non-isomerizing vibrationally excited ground state that relaxes back into the 15E Pfr ground state on a 2.8-ps time scale. Relaxation on a 1-ms timescale results in the loss of red absorbing region, but not blue region, of Lumi-F, which indicates that formation of 15Z Pr occurs on slower timescales.

Scalable and automated CRISPR-based strain engineering using droplet microfluidics.

We present a droplet-based microfluidic system that enables CRISPR-based gene editing and high-throughput screening on a chip. The microfluidic device contains a 10 × 10 element array, and each element contains sets of electrodes for two electric field-actuated operations: electrowetting for merging droplets to mix reagents and electroporation for transformation. This device can perform up to 100 genetic modification reactions in parallel, providing a scalable platform for generating the large number of engineered strains required for the combinatorial optimization of genetic pathways and predictable bioengineering. We demonstrate the system's capabilities through the CRISPR-based engineering of two test cases: (1) disruption of the function of the enzyme galactokinase (galK) in E. coli and (2) targeted engineering of the glutamine synthetase gene (glnA) and the blue-pigment synthetase gene (bpsA) to improve indigoidine production in E. coli.

Meta-analysis of a possible signal of increased mortality associated with cefepime use.

BACKGROUND: On the basis of meta-analyses, concern has been raised regarding a possible signal of increased mortality associated with the use of cefepime versus other beta-lactam antibiotics. To further investigate this possible signal, we accessed findings and data from published and unpublished cefepime clinical trials. METHODS: We performed meta-analyses using trial- and patient-level data from comparative trials. Trial-level analyses were performed using summary data from all patients in the trials, and patient-level analyses were performed on trials for which patient-level data were available. Thirty-day, all-cause mortality was analyzed using the Mantel-Haenszel adjusted risk difference (ARD) method. RESULTS: The trial-level meta-analysis was based on 88 trials (9467 cefepime patients and 8288 comparator patients). The 30-day, all-cause mortality rates were 6.21% (588/9467) for the cefepime patients and 6.00% (497/8288) for comparator patients (ARD per 1000 population, 5.38; 95% confidence interval [CI], -1.53 to 12.28). In the patient-level analysis (35 trials, 5058 cefepime patients, and 3976 comparator patients), 30-day, all-cause mortality rates were 5.63% (285/5058) for cefepime patients and 5.68% (226/3976) for comparator patients (ARD per 1000 population, 4.83; 95% CI, -4.72 to 14.38). A sensitivity analysis based solely on the 24 febrile neutropenia trials did not show a statistically significant increase in mortality with cefepime use (ARD per 1000 population, 9.67; 95% CI, -2.87 to 22.21). CONCLUSIONS: In both trial-level and patient-level meta-analyses, we did not identify a statistically significant increase in mortality among cefepime-treated patients, compared with those treated with other antibacterials.