Assessing the Diagnostic Performance and Clinical Utility of Nasal Methicillin Resistant Staphylococcus aureus PCR Testing in Pediatric Orbital Cellulitis.

Data are limited on the clinical impact of nasal methicillin-resistant Staphylococcus aureus (MRSA) polymerase chain reaction (PCR) testing (nMRSA-PCR) for orbital cellulitis. This two-center, retrospective study demonstrated a negative predictive value of 98.0% and an overall lower use of anti-MRSA antibiotics, without a concomitant increase in hospital readmission.

Neonatal Meningitis Due to Cronobacter sakazakii.

Cronobacter sakazakii is a new emerging foodborne bacterial pathogen associated with fatal infections such as meningitis, necrotizing enterocolitis, and septicemia in neonates. Powdered infant formula milk has been recognized as one of the main transmission vehicles and contaminated sources of this pathogen. Educating parents about the importance of hygienic reconstitution of powdered infant formula, storage practices, and hand hygiene is crucial to reducing the risk of this life-threatening infection. The clinician should be aware of the special considerations for antimicrobial treatment selection as well as further necessary evaluation. Here, we report a case of a twin neonate who presented with C. sakazakii meningitis and septicemia. [Pediatr Ann. 2023;52(11):e430-e433.].

Human parechovirus encephalitis in infants: a retrospective single-center study (2017-2022).

Parechoviruses cause a spectrum of clinical presentations ranging from self-limited to severe encephalitis. In July 2022, state health departments across the USA received an increase in reports of PeV infections among infants. A retrospective cohort study describing the clinical characteristics and outcomes of PeV encephalitis in infants aged < 90 days. Rates of PeV encephalitis were determined based on the number of PeV encephalitis cases out of all meningoencephalitis multiplex polymerase chain reaction panel (MEP) obtained among infants aged < 90 days per year. Out of 2115 infants evaluated for meningoencephalitis, 32 (1.5%) cases of PeV encephalitis were identified. All cases had an absence of pleocytosis and normal protein and glucose levels on CSF analysis. Half of the cases presented with a symptomatic triad (fever, rash, and fussiness). More than one-third of cases (39%) presented with a sepsis-like syndrome, 13% presented with seizures, and 25% were admitted to the pediatric intensive care unit (PICU). MRI of the brain was obtained in four of the cases presented with seizure, all of which demonstrated characteristic radiological findings of the periventricular white matter with frontoparietal predominance and involving the corpus callosum, thalami, and internal and external capsules. Rates of PeV encephalitis varied from year to year, with the highest rates in 2018 and 2022. PeV was the second most detected pathogen in MEP in both 2018 and 2022, and the fifth most detected pathogen in all positive MEP during the study period 2017-2022. CONCLUSION: PeV can cause encephalitis and sepsis-like syndrome in infants, and it should be considered even with normal CSF parameters. Prospective studies are needed to better understand PeV epidemiology and to monitor outbreaks. WHAT IS KNOWN: • PeV is a frequent cause of encephalitis and clinical sepsis in infants in the first 90 days. • Normal CSF parameters in PeV encephalitis and diagnostic importance of MEP to avoid unnecessary prolonged antibiotics and hospitalization.. • Centers for Disease Control and Prevention (CDC) issued a Health Advisory alert in Summer 2022 of uptick PeV encephalitis cases in the USA likely secondary of COVID-19 mitigation measures relaxation, but no comparison with previous years.. WHAT IS NEW: • Knowledge of radiological MRI brain characteristics in PeV encephalitis can be a clue diagnosis. • Knowledge of the biennial seasonality pattern in PeV infection. • PeV was the second most detected pathogen in BIOFIRE ME panel in both 2018 and 2022 in our cohort sample.

Quantifying drug tissue biodistribution by integrating high content screening with deep-learning analysis.

Quantitatively determining in vivo achievable drug concentrations in targeted organs of animal models and subsequent target engagement confirmation is a challenge to drug discovery and translation due to lack of bioassay technologies that can discriminate drug binding with different mechanisms. We have developed a multiplexed and high-throughput method to quantify drug distribution in tissues by integrating high content screening (HCS) with U-Net based deep learning (DL) image analysis models. This technology combination allowed direct visualization and quantification of biologics drug binding in targeted tissues with cellular resolution, thus enabling biologists to objectively determine drug binding kinetics.

Inhibition of IRF5 cellular activity with cell-penetrating peptides that target homodimerization.

The transcription factor interferon regulatory factor 5 (IRF5) plays essential roles in pathogen-induced immunity downstream of Toll-, nucleotide-binding oligomerization domain-, and retinoic acid-inducible gene I-like receptors and is an autoimmune susceptibility gene. Normally, inactive in the cytoplasm, upon stimulation, IRF5 undergoes posttranslational modification(s), homodimerization, and nuclear translocation, where dimers mediate proinflammatory gene transcription. Here, we report the rational design of cell-penetrating peptides (CPPs) that disrupt IRF5 homodimerization. Biochemical and imaging analysis shows that IRF5-CPPs are cell permeable, noncytotoxic, and directly bind to endogenous IRF5. IRF5-CPPs were selective and afforded cell type- and species-specific inhibition. In plasmacytoid dendritic cells, inhibition of IRF5-mediated interferon-α production corresponded to a dose-dependent reduction in nuclear phosphorylated IRF5 [p(Ser462)IRF5], with no effect on pIRF5 levels. These data support that IRF5-CPPs function downstream of phosphorylation. Together, data support the utility of IRF5-CPPs as novel tools to probe IRF5 activation and function in disease.

Loss of MeCP2 in the rat models regression, impaired sociability and transcriptional deficits of Rett syndrome.

Mouse models of the transcriptional modulator Methyl-CpG-Binding Protein 2 (MeCP2) have advanced our understanding of Rett syndrome (RTT). RTT is a 'prototypical' neurodevelopmental disorder with many clinical features overlapping with other intellectual and developmental disabilities (IDD). Therapeutic interventions for RTT may therefore have broader applications. However, the reliance on the laboratory mouse to identify viable therapies for the human condition may present challenges in translating findings from the bench to the clinic. In addition, the need to identify outcome measures in well-chosen animal models is critical for preclinical trials. Here, we report that a novel Mecp2 rat model displays high face validity for modelling psychomotor regression of a learned skill, a deficit that has not been shown in Mecp2 mice. Juvenile play, a behavioural feature that is uniquely present in rats and not mice, is also impaired in female Mecp2 rats. Finally, we demonstrate that evaluating the molecular consequences of the loss of MeCP2 in both mouse and rat may result in higher predictive validity with respect to transcriptional changes in the human RTT brain. These data underscore the similarities and differences caused by the loss of MeCP2 among divergent rodent species which may have important implications for the treatment of individuals with disease-causing MECP2 mutations. Taken together, these findings demonstrate that the Mecp2 rat model is a complementary tool with unique features for the study of RTT and highlight the potential benefit of cross-species analyses in identifying potential disease-relevant preclinical outcome measures.

Fmr1 and Nlgn3 knockout rats: novel tools for investigating autism spectrum disorders.

Animal models are critical for gaining insights into autism spectrum disorder (ASD). Despite their apparent advantages to mice for neural studies, rats have not been widely used for disorders of the human CNS, such as ASD, for the lack of convenient genome manipulation tools. Here we describe two of the first transgenic rat models for ASD, developed using zinc-finger nuclease (ZFN) methodologies, and their initial behavioral assessment using a rapid juvenile test battery. A syndromic and nonsyndromic rat model for ASD were created as two separate knockout rat lines with heritable disruptions in the genes encoding Fragile X mental retardation protein (FMRP) and Neuroligin3 (NLGN3). FMRP, a protein with numerous proposed functions including regulation of mRNA and synaptic protein synthesis, and NLGN3, a member of the neuroligin synaptic cell-adhesion protein family, have been implicated in human ASD. Juvenile subjects from both knockout rat lines exhibited abnormalities in ASD-relevant phenotypes including juvenile play, perseverative behaviors, and sensorimotor gating. These data provide important first evidence regarding the utility of rats as genetic models for investigating ASD-relevant genes.

BET bromodomain proteins mediate downstream signaling events following growth factor stimulation in human lung fibroblasts and are involved in bleomycin-induced pulmonary fibrosis.

Epigenetic alterations, such as histone acetylation, regulate the signaling outcomes and phenotypic responses of fibroblasts after growth factor stimulation. The bromodomain and extra-terminal domain-containing proteins (Brd) bind to acetylated histone residues, resulting in recruitment of components of the transcriptional machinery and subsequent gene transcription. Given the central importance of fibroblasts in tissue fibrosis, this study sought to determine the role of Brd proteins in human lung fibroblasts (LFs) after growth factor stimulation and in the murine bleomycin model of lung fibrosis. Using small interfering RNA against human Brd2 and Brd4 and pharmacologic Brd inhibitors, this study found that Brd2 and Brd4 are essential in mediating the phenotypic responses of LFs downstream of multiple growth factor pathways. Growth factor stimulation of LFs causes increased histone acetylation, association of Brd4 with growth factor-responsive genes, and enhanced transcription of these genes that could be attenuated with pharmacologic Brd inhibitors. Of note, lung fibrosis induced after intratracheal bleomycin challenge in mice could be prevented by pretreatment of animals with pharmacologic inhibitors of Brd proteins. This study is the first demonstration of a role for Brd2 and Brd4 proteins in mediating the responses of LFs after growth factor stimulation and in driving the induction of lung fibrosis in mice in response to bleomycin challenge.

A simple statistical test to infer the causality of target/phenotype correlation from small molecule phenotypic screens.

MOTIVATION: Cell-based phenotypic screens using small molecule inhibitors is an important technology for early drug discovery if the relationship between the disease-related cellular phenotype and inhibitors' biological targets can be determined. However, chemical inhibitors are rightfully believed to be less specific than perturbation by biological agents, such as antibody and small inference RNA. Therefore, it is often a challenge in small molecule phenotypic screening to infer the causality between a particular cellular phenotype and the inactivation of the responsible protein due to the off-target effect of the inhibitors. RESULTS: In this article, we present a Roche in-house effort of screening 746 structurally diverse compounds for their cytotoxicity in HeLa cells measured by high content imaging technology. These compounds were also systematically profiled for the targeted and off-target binding affinity to a panel of 25 pre-selected protein kinases in a cell-free system. In an effort to search for the kinases whose activities are crucial for cell survival, we found that the simple association method such as the chi-square test yields a large number of false positives because the observed cytotoxic phenotype is likely to be the result of promiscuous action of less specific inhibitors instead of true consequence of inactivation of single relevant target. We demonstrated that a stratified categorical data analysis technique such as the Cochran-Mantel-Haenszel test is an effective approach to extract the meaningful biological connection from the spurious correlation resulted from confounding covariates. This study indicates that, empowered by appropriate statistical adjustment, small molecule inhibitor perturbation remains a powerful tool to pin down the relevant biomarker for drug safety and efficacy research. CONTACT: xin.wei@roche.com SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Modifying behavioral phenotypes in Fmr1KO mice: genetic background differences reveal autistic-like responses.

Fragile X syndrome (FXS) is the most common inherited form of intellectual disability in humans. In addition to cognitive impairment, patients may exhibit hyperactivity, attention deficits, social difficulties and anxiety, and autistic-like behaviors. The degree to which patients display these behaviors varies considerably and is influenced by family history, suggesting that genetic modifiers play a role in the expression of behaviors in FXS. Several studies have examined behavior in a mouse model of FXS in which the Fmr1 gene has been ablated. Most of those studies were done in Fmr1 knockout mice on a pure C57BL/6 or FVB strain background. To gain a better understanding of the effects of genetic background on behaviors resulting from the loss of Fmr1 gene expression, we generated F1 hybrid lines from female Fmr1 heterozygous mice on a pure C57BL/6J background bred with male Fmr1 wild-type (WT) mice of various background strains (A/J, DBA/2J, FVB/NJ, 129S1/SvImJ and CD-1). Male Fmr1 knockout and WT littermates from each line were examined in an extensive behavioral test battery. Results clearly indicate that multiple behavioral responses are dependent on genetic background, including autistic-like traits that are present on limited genetic backgrounds. This approach has allowed us to identify improved models for different behavioral symptoms present in FXS including autistic-like traits.

Multiple autism-like behaviors in a novel transgenic mouse model.

Autism spectrum disorder (ASD) diagnoses are behaviorally based with no defined universal biomarkers, occur at a 1:110 ratio in the population, and predominantly affect males compared to females at approximately a 4:1 ratio. One approach to investigate and identify causes of ASD is to use organisms that display abnormal behavioral responses that model ASD-related impairments. This study describes a novel transgenic mouse, MALTT, which was generated using a forward genetics approach. It was determined that the transgene integrated within a non-coding region on the X chromosome. The MALTT line exhibited a complete repertoire of ASD-like behavioral deficits in all three domains required for an ASD diagnosis: reciprocal social interaction, communication, and repetitive or inflexible behaviors. Specifically, MALTT male mice showed deficits in social interaction and interest, abnormalities in pup and juvenile ultrasonic vocalization communications, and exhibited a repetitive stereotypy. Abnormalities were also observed in the domain of sensory function, a secondary phenotype prevalently associated with ASD. Mapping and expression studies suggested that the Fam46 gene family may be linked to the observed ASD-related behaviors. The MALTT line provides a unique genetic model for examining the underlying biological mechanisms involved in ASD-related behaviors.

Valganciclovir for suppression of human herpesvirus-8 replication: a randomized, double-blind, placebo-controlled, crossover trial.

BACKGROUND: Human herpesvirus-8 (HHV-8) replication is critical in the induction and maintenance of Kaposi sarcoma, primary effusion lymphoma, and some cases of Castleman disease. In vitro and observational studies suggest that ganciclovir inhibits HHV-8 replication, but no randomized clinical trials have been conducted. METHODS: A total of 26 men infected with HHV-8 were randomized to receive 8 weeks of valganciclovir administered orally (900 mg once per day) or 8 weeks of placebo administered orally. After a 2-week washout period, participants in each group received the study drug they had not yet taken (either valganciclovir or placebo), for 8 additional weeks. Oral swab samples were collected daily during the study, and HHV-8 and CMV DNA were quantified by real-time PCR. RESULTS: A total of 16 human immunodeficiency virus (HIV)-positive men and 10 HIV-negative men enrolled in and completed the study. Of the 3,439 swab samples that participants had been expected to provide, 3029 (88%) were available for analysis. HHV-8 was detected on 44% of swabs collected from participants who were receiving placebo, compared with 23% of swabs collected from participants who were receiving valganciclovir (relative risk [RR], 0.54 [95% confidence interval {CI}, 0.33-0.90]; P = .02). Valganciclovir reduced oropharyngeal shedding of cytomegalovirus by 80% (RR, 0.20 [95% CI, 0.08-0.48]; P < .001). Shedding of HHV-8 and shedding of cytomegalovirus were independent. Hematologic, renal, or hepatic toxicities were no more common among participants who received the active drug, compared with those who received placebo, though participants who received valganciclovir reported more days of diarrhea. CONCLUSIONS: Valganciclovir administered orally once per day is well tolerated and significantly reduces the frequency and quantity of HHV-8 replication.

Regulation of the extracellular matrix remodeling by lutein in dermal fibroblasts, melanoma cells, and ultraviolet radiation exposed fibroblasts.

With aging and cancer there is increased expression or activity of matrix metalloproteinases (MMPs) that degrade and remodel the structural extracellular matrix (ECM). In addition, exposure of skin to ultraviolet (UV) radiation (photoaging) leads to loss of cell viability, membrane damage, and deposition of excessive elastotic material. Lutein has antioxidant, anti-inflammatory, photoprotective, and anti-carcinogenic properties. The goal of this research was to investigate lutein's anti-aging and anti-carcinogenic effects via the regulation of the extracellular matrix remodeling. To this purpose, the effects of lutein on the expression of MMPs and their inhibitors (TIMPs, tissue inhibitors of metalloproteinases) in dermal fibroblasts (intrinsic aging) and melanoma cells were examined. Further, for lutein's photoprotective effects, the regulation of cell viability, membrane integrity, and elastin expression in the non-irradiated, and UVA or UVB radiation exposed fibroblasts were analyzed. Lutein significantly inhibited MMP-1 expression, transcriptionally, and MMP-2 protein levels in dermal fibroblasts, without altering TIMPs expression. It significantly inhibited MMP-1 expression in melanoma cells while stimulating TIMP-2. Lutein did not alter fibroblast or melanoma cell viability or membrane integrity. In ultraviolet radiation exposed fibroblasts, lutein improved cell viability, membrane integrity and inhibited elastin expression, though more significantly in the UVB exposed fibroblasts. In summary, the mechanism to lutein's anti-aging and anti-carcinogenic effects include the inhibition of MMP to TIMP ratio in dermal fibroblasts and melanoma cells, and the inhibition of cell loss, membrane damage and elastin expression in ultraviolet radiation exposed fibroblasts.

Comparison of oligonucleotide ligation assay and consensus sequencing for detection of drug-resistant mutants of human immunodeficiency virus type 1 in peripheral blood mononuclear cells and plasma.

Drug-resistant mutants of human immunodeficiency virus type 1 (HIV-1) recede below the limit of detection of most assays applied to plasma when selective pressure is altered due to changes in antiretroviral treatment (ART). Viral variants with different mutations are selected by the new ART when replication is not suppressed or wild-type variants with greater replication fitness outgrow mutants following the cessation of ART. Mutants selected by past ART appear to persist in reservoirs even when not detected in the plasma, and when conferring cross-resistance they can compromise the efficacy of novel ART. Oligonucleotide ligation assay (OLA) of virus in plasma and peripheral blood mononuclear cells (PBMC) was compared to consensus sequence dideoxynucleotide chain terminator sequencing for detection of 91 drug resistance mutations that had receded below the limit of detection by sequencing of plasma. OLA of plasma virus detected 27.5% (95% confidence interval [CI], 19 to 39%) of mutant genotypes; consensus sequencing of the PBMC amplicon from the same specimen detected 23.1% (95% CI, 14 to 34%); and OLA of PBMC detected 53.8% (95% CI, 44 to 64%). These data suggest that concentrations of drug-resistant mutants were greater in PBMC than in plasma after changes in ART and indicate that the OLA was more sensitive than consensus sequencing in detecting low levels of select drug-resistant mutants.

Rapid and sensitive oligonucleotide ligation assay for detection of mutations in human immunodeficiency virus type 1 associated with high-level resistance to protease inhibitors.

A sensitive, specific, and high-throughput oligonucleotide ligation assay (OLA) for the detection of genotypic human immunodeficiency virus type 1 (HIV-1) resistance to Food and Drug Administration-approved protease inhibitors was developed and evaluated. This ligation-based assay uses differentially modified oligonucleotides specific for wild-type or mutant sequences, allowing sensitive and simple detection of both genotypes in a single well of a microtiter plate. Oligonucleotides were designed to detect primary mutations associated with high-level resistance to amprenavir, nelfinavir, indinavir, ritonavir, saquinavir, and lopinavir, including amino acid substitutions D30N, I50V, V82A/S/T, I84V, N88D, and L90M. Plasma HIV-1 RNA from 54 infected patients was amplified by reverse transcription-PCR and sequenced by using dideoxynucleotide chain terminators for evaluation of mutations associated with drug resistance. These same amplicons were genotyped by the OLA at positions 30, 50, 82, 88, 84, and 90 for a total of 312 codons. The sensitivity of detection of drug-resistant genotypes was 96.7% (87 of 90 mutant codons) in the OLA compared to 92.2% (83 of 90) in consensus sequencing, presumably due to the increased sensitivity of the OLA. The OLA detected genetic subpopulations more often than sequencing, detecting 30 mixtures of mutant and wild-type sequences and two mixtures of drug-resistant sequences compared to 15 detected by DNA sequencing. Reproducible and semiquantitative detection of the mutant and the wild-type genomes by the OLA was observed by analysis of wild-type and mutant plasmid mixtures containing as little as 5% of either genotype in a background of the opposite genome. This rapid, simple, economical, and highly sensitive assay provides a practical alternative to dideoxy sequencing for genotypic evaluation of HIV-1 resistance to antiretrovirals.

Three-dimensional solution structure of the calcium-signaling protein apo-S100A1 as determined by NMR.

S100A1, a member of the S100 protein family, is an EF-hand containing Ca(2+)-binding protein (93 residues per subunit) with noncovalent interactions at its dimer interface. Each subunit of S100A1 has four alpha-helices and a small antiparallel beta-sheet consistent with two helix-loop-helix calcium-binding domains [Baldiserri et al. (1999) J. Biomol. NMR 14, 87-88]. In this study, the three-dimensional structure of reduced apo-S100A1 was determined by NMR spectroscopy using a total of 2220 NOE distance constraints, 258 dihedral angle constraints, and 168 backbone hydrogen bond constraints derived from a series of 2D, 3D, and 4D NMR experiments. The final structure was found to be globular and compact with the four helices in each subunit aligning to form a unicornate-type four-helix bundle. Intermolecular NOE correlations were observed between residues in helices 1 and 4 from one subunit to residues in helices 1' and 4' of the other subunit, respectively, consistent with the antiparallel alignment of the two subunits to form a symmetric X-type four-helix bundle as found for other members of the S100 protein family. Because of the similarity of the S100A1 dimer interface to that found for S100B, it was possible to calculate a model of the S100A1/B heterodimer. This model is consistent with a number of NMR chemical shift changes observed when S100A1 is titrated into a sample of (15)N-labeled S100B. Helix 3 (and 3') of S100A1 was found to have an interhelical angle of -150 degrees with helix 4 (and 4') in the apo state. This crossing angle is quite different (>50 degrees ) from that typically found in other EF-hand containing proteins such as apocalmodulin and apotroponin C but more similar to apo-S100B, which has an interhelical angle of -166 degrees. As with S100B, it is likely that the second EF-hand of apo-S100A1 reorients dramatically upon the addition of Ca(2+), which can explain the Ca(2+) dependence that S100A1 has for binding several of its biological targets.