Assessing the safety profile of voriconazole use in suspected COVID-19-associated pulmonary aspergillosis-a two-centre observational study.

The decision to use voriconazole for suspected COVID-19-associated pulmonary aspergillosis (CAPA) is based on clinical judgement weighed against concerns about its potential toxicity. We assessed the safety profile of voriconazole for patients with suspected CAPA by conducting a retrospective study of patients across two intensive care units. We compared changes in any liver enzymes or bilirubin and any new or increasing corrected QT interval (QTc) prolongation following voriconazole use to patient baseline to indicate possible drug effect. In total, 48 patients with presumed CAPA treated with voriconazole were identified. Voriconazole therapy was administered for a median of 8 days (interquartile range [IQR] 5-22) and the median level was 1.86 mg/L (IQR 1.22-2.94). At baseline, 2% of patients had a hepatocellular injury profile, 54% had a cholestatic injury profile, and 21% had a mixed injury profile. There were no statistically significant changes in liver function tests over the first 7 days after voriconazole initiation. At day 28, there was a significant increase in alkaline phospahte only (81-122 U/L, P = 0.006), driven by changes in patients with baseline cholestatic injury. In contrast, patients with baseline hepatocellular or mixed injury had a significant decrease in alanine transaminase and aspartate transaminase. Baseline QTc was 437 ms and remained unchanged after 7 days of voriconazole therapy even after sensitivity analysis for concomitantly administered QT prolonging agents. Therefore, at the doses used in this study, we did not detect evidence of significant liver or cardiac toxicity related to voriconazole use. Such information can be used to assist clinicians in the decision to initiate such treatment.

Our study did not show significant voriconazole-related liver or cardiac side effects in a critically ill cohort of patients with suspected COVID-19-associated pulmonary aspergillosis. These findings may allay specific clinician concerns when commencing therapy for such patients.

Neutrophil kinetics and function after major trauma: A systematic review.

BACKGROUND: Immune dysfunction following major traumatic injury is complex and strongly associated with significant morbidity and mortality through the development of multiple organ dysfunction syndrome (MODS), persistent inflammation, immunosuppression, and catabolism syndrome and sepsis. Neutrophils are thought to be a pivotal mediator in the development of immune dysfunction. AIM: To provide a review with a systematic approach of the recent literature describing neutrophil kinetics and functional changes after major trauma in humans and discuss hypotheses as to the mechanisms of the observed neutrophil dysfunction in this setting. METHODS: Medline, Embase and PubMed were searched on January 15, 2021. Papers were screened by two reviewers and those included had their reference list hand searched for additional papers of interest. Inclusion criteria were adults > 18 years old, with an injury severity score > 12 requiring admission to an intensive care unit. Papers that analysed major trauma patients as a subgroup were included. RESULTS: Of 107 papers screened, 48 were included in the review. Data were heterogeneous and most studies had a moderate to significant risk of bias owing to their observational nature and small sample sizes. Key findings included a persistently elevated neutrophil count, stereotyped alterations in cell-surface markers of activation, and the elaboration of heterogeneous and immunosuppressive populations of cells in the circulation. Some of these changes correlate with clinical outcomes such as MODS and secondary infection. Neutrophil phenotype remains a promising avenue for the development of predictive markers for immune dysfunction. CONCLUSION: Understanding of neutrophil phenotypes after traumatic injury is expanding. A greater emphasis on incorporating functional and clinically significant markers, greater uniformity in study design and assessment of extravasated neutrophils may facilitate risk stratification in patients affected by major trauma.

Haemophilus influenzae type b necrotizing fasciitis in an adult with common variable immunodeficiency.

Necrotizing fasciitis of an extremity due to Haemophilus influenzae is exceptionally uncommon in adults, particularly since the advent of widespread vaccination with conjugated H. influenzae type b (Hib). We report a previously vaccinated, 39-year-old male with a history of common variable immunodeficiency (CVID), poorly compliant with intravenous immunoglobulin (IVIG) therapy, who required emergent treatment for left leg necrotizing fasciitis. The patient was initially treated with piperacillin-tazobactam, vancomycin, and clindamycin, in tandem with surgical debridement and wash-out. The patient responded well and completed a 2-week course of ceftriaxone following blood culture results that demonstrated beta-lactamase-positive Hib. This is the fifth documented case of necrotizing fasciitis caused by H. influenzae, and the first affecting an adult with prior Hib vaccination. This case highlights the importance of IVIG compliance for CVID patients and advocates considering encapsulated organisms as part of the differential diagnosis for severe skin and soft tissue infections in this patient population.

La fasciite nécrosante d'une extrémité, causée par l'Haemophilus influenzae, est d'une extrême rareté chez les adultes, particulièrement depuis l'adoption de la vaccination généralisée par le vaccin conjugué contre l'H. influenzae de type b (Hib). Les auteurs signalent le cas d'un homme de 39 ans déjà vacciné ayant une histoire d'hypogammaglobulinémie à expression variable, qui adhérait peu au traitement aux immunoglobulines intraveineuses et qui a dû recevoir un traitement d'urgence contre une fasciite nécrosante de la jambe gauche. Il a d'abord reçu du pipéracilline-tazobactam, de la vancomycine et de la clindamycine, conjointement avec un débridement chirurgical et un lavage. Le patient a bien répondu et a terminé un cycle de cetriaxone de deux semaines après une hémoculture qui a révélé un Hib positif à la bêta-lactamase. Il s'agit du cinquième cas démontré de fasciite nécrosante causée par l'H. influenzae, et le premier touchant un adulte déjà vacciné contre le Hib. Ce cas fait ressortir l'importance de respecter le traitement aux immunoglobulines intraveineuses en cas d'hypogammaglobulinémie à expression variable et démontre l'intérêt d'envisager des organismes encapsulés dans le diagnostic différentiel de graves infections de la peau et des tissus mous auprès de cette population de patients.

Age-related loss of hepatic Nrf2 protein homeostasis: Potential role for heightened expression of miR-146a.

Nrf2 regulates the expression of numerous anti-oxidant, anti-inflammatory, and metabolic genes. We observed that, paradoxically, Nrf2 protein levels decline in the livers of aged rats despite the inflammatory environment evident in that organ. To examine the cause(s) of this loss, we investigated the age-related changes in Nrf2 protein homeostasis and activation in cultured hepatocytes from young (4-6 months) and old (24-28 months) Fischer 344 rats. While no age-dependent change in Nrf2 mRNA levels was observed (p>0.05), Nrf2 protein content, and the basal and anetholetrithione (A3T)-induced expression of Nrf2-dependent genes were attenuated with age. Conversely, overexpression of Nrf2 in cells from old animals reinstated gene induction. Treatment with A3T, along with bortezomib to inhibit degradation of existing protein, caused Nrf2 to accumulate significantly in cells from young animals (p<0.05), but not old, indicating a lack of new Nrf2 synthesis. We hypothesized that the loss of Nrf2 protein synthesis with age may partly stem from an age-related increase in microRNA inhibition of Nrf2 translation. Microarray analysis revealed that six microRNAs significantly increase >2-fold with age (p<0.05). One of these, miRNA-146a, is predicted to bind Nrf2 mRNA. Transfection of hepatocytes from young rats with a miRNA-146a mimic caused a 55% attenuation of Nrf2 translation that paralleled the age-related loss of Nrf2. Overall, these results provide novel insights for the age-related decline in Nrf2 and identify new targets to maintain Nrf2-dependent detoxification with age.

Lipoic acid entrains the hepatic circadian clock and lipid metabolic proteins that have been desynchronized with advanced age.

It is well established that lipid metabolism is controlled, in part, by circadian clocks. However, circadian clocks lose temporal precision with age and correlates with elevated incidence in dyslipidemia and metabolic syndrome in older adults. Because our lab has shown that lipoic acid (LA) improves lipid homeostasis in aged animals, we hypothesized that LA affects the circadian clock to achieve these results. We fed 24 month old male F344 rats a diet supplemented with 0.2% (w/w) LA for 2 weeks prior to sacrifice and quantified hepatic circadian clock protein levels and clock-controlled lipid metabolic enzymes. LA treatment caused a significant phase-shift in the expression patterns of the circadian clock proteins Period (Per) 2, Brain and Muscle Arnt-Like1 (BMAL1), and Reverse Erythroblastosis virus (Rev-erb) ß without altering the amplitude of protein levels during the light phase of the day. LA also significantly altered the oscillatory patterns of clock-controlled proteins associated with lipid metabolism. The level of peroxisome proliferator-activated receptor (PPAR) α was significantly increased and acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) were both significantly reduced, suggesting that the LA-supplemented aged animals are in a catabolic state. We conclude that LA remediates some of the dyslipidemic processes associated with advanced age, and this mechanism may be at least partially through entrainment of circadian clocks.

R-α-lipoic acid does not reverse hepatic inflammation of aging, but lowers lipid anabolism, while accentuating circadian rhythm transcript profiles.

To determine the effects of age and lipoic acid supplementation on hepatic gene expression, we fed young (3 mo) and old (24 mo) male Fischer 344 rats a diet with or without 0.2% (wt/wt) R-α-lipoic acid (LA) for 2 wk. Total RNA isolated from liver tissue was analyzed by Affymetrix microarray to examine changes in transcriptional profiles. Results showed elevated proinflammatory gene expression in the aging liver and evidence for increased immune cell activation and tissue remodeling, together representing 45% of the age-related transcriptome changes. In addition, age-related increases in transcripts of genes related to fatty acid, triglyceride, and cholesterol synthesis, including acetyl-CoA carboxylase-ß (Acacb) and fatty acid synthase (Fasn), were observed. Supplementation of old animals with LA did not reverse the necroinflammatory phenotype but, intriguingly, altered the expression of genes governing circadian rhythm. Most notably, Arntl, Npas2, and Per changed in a coordinated manner with respect to rhythmic transcription. LA further caused a decrease in transcripts of several bile acid and lipid synthesis genes, including Acacb and Fasn, which are regulated by first-order clock transcription factors. Similar effects of LA supplementation on bile acid and lipid synthesis genes were observed in young animals. Transcript changes of lipid metabolism genes were corroborated by a decrease in FASN and ACC protein levels. We conclude that advanced age is associated with a necroinflammatory phenotype and increased lipid synthesis, while chronic LA supplementation influences hepatic genes associated with lipid and energy metabolism and circadian rhythm, regardless of age.

(R)-α-Lipoic acid treatment restores ceramide balance in aging rat cardiac mitochondria.

Inflammation results in heightened mitochondrial ceramide levels, which cause electron transport chain dysfunction, elevates reactive oxygen species, and increases apoptosis. As mitochondria in aged hearts also display many of these characteristics, we hypothesized that mitochondrial decay stems partly from an age-related ceramidosis that heretofore has not been recognized for the heart. Intact mitochondria or their purified inner membranes (IMM) were isolated from young (4-6 mo) and old (26-28 mo) rats and analyzed for ceramides by LC-MS/MS. Results showed that ceramide levels increased by 32% with age and three ceramide isoforms, found primarily in the IMM (e.g. C(16)-, C(18)-, and C(24:1)-ceramide), caused this increase. The ceramidosis may stem from enhanced hydrolysis of sphingomyelin, as neutral sphingomyelinase (nSMase) activity doubled with age but with no attendant change in ceramidase activity. Because (R)-α-lipoic acid (LA) improves many parameters of cardiac mitochondrial decay in aging and lowers ceramide levels in vascular endothelial cells, we hypothesized that LA may limit cardiac ceramidosis and thereby improve mitochondrial function. Feeding LA [0.2%, w/w] to old rats for two weeks prior to mitochondrial isolation reversed the age-associated decline in glutathione levels and concomitantly improved Complex IV activity. This improvement was associated with lower nSMase activity and a remediation in mitochondrial ceramide levels. In summary, LA treatment lowers ceramide levels to that seen in young rat heart mitochondria and restores Complex IV activity which otherwise declines with age.

Seasonality and vertical structure of microbial communities in an ocean gyre.

Vertical, seasonal and geographical patterns in ocean microbial communities have been observed in many studies, but the resolution of community dynamics has been limited by the scope of data sets, which are seldom up to the task of illuminating the highly structured and rhythmic patterns of change found in ocean ecosystems. We studied vertical and temporal patterns in the microbial community composition in a set of 412 samples collected from the upper 300 m of the water column in the northwestern Sargasso Sea, on cruises between 1991 and 2004. The region sampled spans the extent of deep winter mixing and the transition between the euphotic and the upper mesopelagic zones, where most carbon fixation and reoxidation occurs. A bioinformatic pipeline was developed to de-noise, normalize and align terminal restriction fragment length polymorphism (T-RFLP) data from three restriction enzymes and link T-RFLP peaks to microbial clades. Non-metric multidimensional scaling statistics resolved three microbial communities with distinctive composition during seasonal stratification: a surface community in the region of lowest nutrients, a deep chlorophyll maximum community and an upper mesopelagic community. A fourth microbial community was associated with annual spring blooms of eukaryotic phytoplankton that occur in the northwestern Sargasso Sea as a consequence of winter convective mixing that entrains nutrients to the surface. Many bacterial clades bloomed in seasonal patterns that shifted with the progression of stratification. These richly detailed patterns of community change suggest that highly specialized adaptations and interactions govern the success of microbial populations in the oligotrophic ocean.

Analysis of the retrovirus capsid interdomain linker region.

In structural studies, the retrovirus capsid interdomain linker region has been shown as a flexible connector between the CA N-terminal domain and its C-terminal domain. To analyze the function of the linker region, we have examined the effects of three Moloney murine leukemia virus (M-MuLV) capsid linker mutations/variations in vivo, in the context of the full-length M-MuLV structural precursor protein (PrGag). Two mutations, A1SP and A5SP, respectively, inserted three and seven additional codons within the linker region to test the effects of increased linker lengths. The third variant, HIV/Mo, represented a chimeric HIV-1/M-MuLV PrGag protein, fused at the linker region. When expressed in cells, the three variants reduced the efficiency of virus particle assembly, with PrGag proteins and particles accumulating at the cellular plasma membranes. Although PrGag recognition of viral RNA was not impaired, the capsid linker variant particles were abnormal, with decreased stabilities, anomalous densities, and aberrant multiple lobed and tubular morphologies. Additionally, rather than crosslinking as PrGag dimers, particle-associated A1SP, A5SP, and HIV/Mo proteins showed an increased propensity to crosslink as trimers. Our results suggest that a wild-type retrovirus capsid linker region is required for the proper alignment of capsid protein domains.

Retrovirus capsid protein assembly arrangements.

During retrovirus particle assembly and morphogenesis, the retrovirus structural (Gag) proteins organize into two different arrangements: an immature form assembled by precursor Gag (PrGag) proteins; and a mature form, composed of proteins processed from PrGag. Central to both Gag protein arrangements is the capsid (CA) protein, a domain of PrGag, which is cleaved from the precursor to yield a mature Gag protein composed of an N-terminal domain (NTD), a flexible linker region, and a C-terminal domain (CTD). Because Gag interactions have proven difficult to examine in virions, a number of investigations have focused on the analysis of structures assembled in vitro. We have used electron microscope (EM) image reconstruction techniques to examine assembly products formed by two different CA variants of both human immunodeficiency virus type 1 (HIV-1) and the Moloney murine leukemia virus (M-MuLV). Interestingly, two types of hexameric protein arrangements were observed for each virus type. One organizational scheme featured hexamers composed of putative NTD dimer subunits, with sharing of subunits between neighbor hexamers. The second arrangement used apparent NTD monomers to coordinate hexamers, involved no subunit sharing, and employed putative CTD interactions to connect hexamers. Conversion between the two assembly forms may be achieved by making or breaking the proposed symmetric NTD dimer contacts in a process that appears to mimic viral morphogenesis.

Hantavirus nucleocapsid protein coiled-coil domains.

The nucleocapsid (N) proteins of hantaviruses such as the Sin Nombre virus (SNV) bind to membranes and viral RNAs, associate with transcription and replication complexes, and oligomerize during the process of virus assembly. N proteins trimerize in vitro and in vivo, and associate via assembly domains at their amino- and carboxyl-terminal ends. Because structure prediction algorithms suggested that N protein residues 3-75 form two coiled-coil motifs separated by an intervening kink or turn sequence, we examined the properties of peptides representing SNV N protein residues 3-35, 43-75, and 3-75. Of the three peptides, N-(3-35) assembled coiled-coil oligomers only at high concentration and low temperature. In contrast, N-(43-75) efficiently trimerized at low concentration, implying that it carries a coiled-coil trigger sequence. Interestingly, while the longer peptide, N-(3-75), assembled dimers and/or trimers at high concentration, at low concentration it appeared to adopt an intramolecular helix-turn-helix conformation. These results suggest that N protein oligomerization involves the bundling of intramolecular antiparallel coils or a conformational switch from intra- to intermolecular coiled-coils.