Inhibition of DXR in the MEP pathway with lipophilic N-alkoxyaryl FR900098 analogs.

In Mycobacterium tuberculosis (Mtb) and Plasmodium falciparum (Pf), the methylerythritol phosphate (MEP) pathway is responsible for isoprene synthesis. This pathway and its products are vital to bacterial/parasitic metabolism and survival, and represent an attractive set of drug targets due to their essentiality in these pathogens but absence in humans. The second step in the MEP pathway is the conversion of 1-deoxy-d-xylulose-5-phosphate (DXP) to MEP and is catalyzed by 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR). Natural products fosmidomycin and FR900098 inhibit DXR, but are too polar to reach the desired target inside some cells, such as Mtb. Synthesized FR900098 analogs with lipophilic substitution in the position α to the phosphorous atom showed promise, resulting in increased activity against Mtb and Pf. Here, an α substitution, consisting of a 3,4-dichlorophenyl substituent, in combination with various O-linked alkylaryl substituents on the hydroxamate moiety is utilized in the synthesis of a novel series of FR900098 analogs. The purpose of the O-linked alkylaryl substituents is to further enhance DXR inhibition by extending the structure into the adjacent NADPH binding pocket, blocking the binding of both DXP and NADPH. Of the initial O-linked alkylaryl substituted analogs, compound 6e showed most potent activity against Pf parasites at 3.60 µM. Additional compounds varying the phenyl ring of 6e were synthesized. The most potent phosphonic acids, 6l and 6n, display nM activity against PfDXR and low µM activity against Pf parasites. Prodrugs of these compounds were less effective against Pf parasites but showed modest activity against Mtb cells. Data from this series of compounds suggests that this combination of substituents can be advantageous in designing a new generation of antimicrobials.

Within-host influenza viral diversity in the pediatric population as a function of age, vaccine, and health status.

Seasonal influenza virus predominantly evolves through antigenic drift, marked by the accumulation of mutations at antigenic sites. Because of antigenic drift, influenza vaccines are frequently updated, though their efficacy may still be limited due to strain mismatches. Despite the high levels of viral diversity observed across populations, most human studies reveal limited intrahost diversity, leaving the origin of population-level viral diversity unclear. Previous studies show host characteristics, such as immunity, might affect within-host viral evolution. Here we investigate influenza A viral diversity in children aged between 6 months and 18 years. Influenza virus evolution in children is less well characterized than in adults, yet may be associated with higher levels of viral diversity given the lower level of pre-existing immunity and longer durations of infection in children. We obtained influenza isolates from banked influenza A-positive nasopharyngeal swabs collected at the Children's Hospital of Philadelphia during the 2017-18 influenza season. Using next-generation sequencing, we evaluated the population of influenza viruses present in each sample. We characterized within-host viral diversity using the number and frequency of intrahost single-nucleotide variants (iSNVs) detected in each sample. We related viral diversity to clinical metadata, including subjects' age, vaccination status, and comorbid conditions, as well as sample metadata such as virus strain and cycle threshold. Consistent with previous studies, most samples contained low levels of diversity with no clear association between the subjects' age, vaccine status, or health status. Further, there was no enrichment of iSNVs near known antigenic sites. Taken together, these findings are consistent with previous observations that the majority of intrahost influenza virus infection is characterized by low viral diversity without evidence of diversifying selection.

Breath biomarkers of pediatric malaria: reproducibility and response to antimalarial therapy.

BACKGROUND: Many insect-borne pathogens appear to manipulate the odors of their hosts in ways that influence vector behaviors. In our prior work, we identified characteristic changes in volatile emissions of cultured Plasmodium falciparum parasites in vitro and during natural human falciparum malaria. In the current study, we prospectively evaluate the reproducibility of these findings in an independent cohort of children in Blantyre, Malawi. METHODS: We enrolled febrile children under evaluation for malaria and collected breath from children with and without malaria, as well as healthy controls. Using gas-chromatography/mass spectrometry, we characterized breath volatiles associated with malaria. By repeated sampling of children with malaria before and after antimalarial use, we determined how breath profiles respond to treatment. In addition, we investigated the stage-specificity of biomarkers through correlation with asexual and sexual stage parasitemia. RESULTS: Our data provide robust evidence that P. falciparum infection leads to specific, reproducible changes in breath compounds. While no individual compound served as adequate classifier in isolation, selected volatiles together yielded high sensitivity for diagnosis of malaria. Overall, the results of our predictive models suggest the presence of volatile signatures that reproducibly predict malaria infection status and determine response to therapy, even in cases of low parasitemia.

Improving access to exome sequencing in a medically underserved population through the Texome Project.

PURPOSE: Genomic medicine can end diagnostic odysseys for patients with complex phenotypes; however, limitations in insurance coverage and other systemic barriers preclude individuals from accessing comprehensive genetics evaluation and testing. METHODS: The Texome Project is a 4-year study that reduces barriers to genomic testing for individuals from underserved and underrepresented populations. Participants with undiagnosed, rare diseases who have financial barriers to obtaining exome sequencing (ES) clinically are enrolled in the Texome Project. RESULTS: We highlight the Texome Project process and describe the outcomes of the first 60 ES results for study participants. Participants received a genetic evaluation, ES, and return of results at no cost. We summarize the psychosocial or medical implications of these genetic diagnoses. Thus far, ES provided molecular diagnoses for 18 out of 60 (30%) of Texome participants. Plus, in 11 out of 60 (18%) participants, a partial or probable diagnosis was identified. Overall, 5 participants had a change in medical management. CONCLUSION: To date, the Texome Project has recruited a racially, ethnically, and socioeconomically diverse cohort. The diagnostic rate and medical impact in this cohort support the need for expanded access to genetic testing and services. The Texome Project will continue reducing barriers to genomic care throughout the future study years.

High Target Homology Does Not Guarantee Inhibition: Aminothiazoles Emerge as Inhibitors of Plasmodium falciparum.

In this study, we identified three novel compound classes with potent activity against Plasmodium falciparum, the most dangerous human malarial parasite. Resistance of this pathogen to known drugs is increasing, and compounds with different modes of action are urgently needed. One promising drug target is the enzyme 1-deoxy-d-xylulose-5-phosphate synthase (DXPS) of the methylerythritol 4-phosphate (MEP) pathway for which we have previously identified three active compound classes against Mycobacterium tuberculosis. The close structural similarities of the active sites of the DXPS enzymes of P. falciparum and M. tuberculosis prompted investigation of their antiparasitic action, all classes display good cell-based activity. Through structure-activity relationship studies, we increased their antimalarial potency and two classes also show good metabolic stability and low toxicity against human liver cells. The most active compound 1 inhibits the growth of blood-stage P. falciparum with an IC50 of 600 nM. The results from three different methods for target validation of compound 1 suggest no engagement of DXPS. All inhibitor classes are active against chloroquine-resistant strains, confirming a new mode of action that has to be further investigated.

Metabolomic and Immunologic Discriminators of MIS-C at Emergency Room Presentation.

Multisystem Inflammatory Syndrome in Childhood (MIS-C) follows SARS-CoV-2 infection and frequently leads to intensive care unit admission. The inability to rapidly discriminate MIS-C from similar febrile illnesses delays treatment and leads to misdiagnosis. To identify diagnostic discriminators at the time of emergency department presentation, we enrolled 104 children who met MIS-C screening criteria, 14 of whom were eventually diagnosed with MIS-C. Before treatment, we collected breath samples for volatiles and peripheral blood for measurement of plasma proteins and immune cell features. Clinical and laboratory features were used as inputs for a machine learning model to determine diagnostic importance. MIS-C was associated with significant changes in breath volatile organic compound (VOC) composition as well as increased plasma levels of secretory phospholipase A2 (PLA2G2A) and lipopolysaccharide binding protein (LBP). In an integrated model of all analytes, the proportion of TCRVß21.3+ non-naive CD4 T cells expressing Ki-67 had a high sensitivity and specificity for MIS-C, with diagnostic accuracy further enhanced by low sodium and high PLA2G2A. We anticipate that accurate diagnosis will become increasingly difficult as MIS-C becomes less common. Clinical validation and application of this diagnostic model may improve outcomes in children presenting with multisystem febrile illnesses.

Intrafamilial phenotypic heterogeneity in siblings with pseudohypoparathyroidism 1B due to maternal STX16 deletion.

OBJECTIVES: Pseudohypoparathyroidism (PHP1B) is most commonly caused by epigenetic defects resulting in loss of methylation at the GNAS locus, although deletions of STX16 leading to GNAS methylation abnormalities have been previously reported. The phenotype of this disorder is variable and can include hormonal resistances and severe infantile obesity with hyperphagia. A possible time relationship between the onset of obesity and endocrinopathies has been previously reported but remains unclear. Understanding of the condition's natural history is limited, partly due to a scarcity of literature, especially in children. CASE PRESENTATION: We report three siblings with autosomal dominant PHP1B caused by a deletion in STX16 who presented with early childhood onset PTH-resistance with normocalcemia with a progressive nature, accompanied by TSH-resistance and severe infantile obesity with hyperphagia in some, not all of the affected individuals. CONCLUSIONS: PHP1B from a STX16 deletion displays intrafamilial phenotypic variation. It is a novel cause of severe infantile obesity, which is not typically included in commercially available gene panels but must be considered in the genetic work-up. Finally, it does not seem to have a clear time relationship between the onset of obesity and hormonal resistance.

Diagnosis and management of facial nerve palsy secondary to granulomatosis with polyangiitis - A systematic review.

OBJECTIVE: Granulomatosis with polyangiitis is associated with otolaryngologic complaints in 70-95 % of cases, with the most common being serous otitis media. In rare cases, patients may experience facial nerve palsy in conjunction with otologic or nasal symptoms; and, often, initially present to an otolaryngologist. It is important for healthcare professionals to be able to recognize the nuisances of facial nerve palsy as a potential presentation of granulomatosis with polyangiitis. STUDY DESIGN: Systematic review. METHODS: Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Protocol, PubMed and MED-LINE Databases were queried for articles published from January 2007 to December 2022 describing facial nerve palsy in the context of Granulomatosis with polyangiitis, formerly known as Wegener's Granulomatosis. The keywords included "facial nerve palsy", "facial palsy", "granulomatosis with polyangiitis", "Wegener's granulomatosis", "ANCA positive" in the title/abstract. All full-text articles available in English were screened, including single case presentations. Abstracts, commentaries, and publications deemed outside the scope of our study aims were excluded from review. After removal of duplicate articles, a total of 85 articles were screened. After applying inclusion and exclusion criteria, 14 articles were included in the review. RESULTS: There were a total of 28 reports of facial nerve palsy in the literature in patients who were eventually diagnosed with granulomatosis with polyangiitis. The patients' ages ranged from 14 to 68 years old. None of the patients had been previously diagnosed with GPA, and a majority of them presented initially with other otologic symptoms. Hearing loss was reported in 24 patients (86 %), otalgia was present in 11 patients (39 %), and otorrhea was present in 6 patients (21 %). Bilateral facial paralysis was reported in 10 patients in the literature (36 %). In total, 16 patients underwent surgery for facial paralysis: 6 tympanomastoidectomies, 4 mastoidectomies, 2 explorative tympanotomies. Surgery was generally considered ineffective in resolving facial weakness. All patients ended up receiving some combination of steroids and immunosuppressant, most commonly prednisolone and cyclophosphamide or rituximab, which was eventually transitioned to azathioprine for maintenance. Unlike auditory thresholds, which remained decreased in two patients, all patients recovered facial function following appropriate medical treatment of their vasculitis. CONCLUSIONS: Facial nerve paralysis in patients with granulomatosis with polyangiitis is a rare but treatable phenomenon. In patients with intractable otitis media, unresolving facial palsy, or a combination of otologic issues, it is important to consider GPA as a possible source. The prognosis for facial function appears to be excellent in patients who undergo appropriate treatment for vasculitis, but further studies are needed for confirmation.

Volatile profiling distinguishes Streptococcus pyogenes from other respiratory streptococcal species.

Sore throat is one of the most common complaints encountered in the ambulatory clinical setting. Rapid, culture-independent diagnostic techniques that do not rely on pharyngeal swabs would be highly valuable as a point-of-care strategy to guide outpatient antibiotic treatment. Despite the promise of this approach, efforts to detect volatiles during oropharyngeal infection have yet been limited. In our research study, we sought to evaluate for specific bacterial volatile organic compounds (VOC) biomarkers in isolated cultures in vitro, in order to establish proof-of-concept prior to initial clinical studies of breath biomarkers. A particular challenge for the diagnosis of pharyngitis due to Streptococcus pyogenes is the likelihood that many metabolites may be shared by S. pyogenes and other related oropharyngeal colonizing bacterial species. Therefore, we evaluated whether sufficient metabolic differences are present, which distinguish the volatile metabolome of Group A streptococci from other streptococcal species that also colonize the respiratory mucosa, such as Streptococcus pneumoniae and Streptococcus intermedius. In this work, we identified 27 discriminatory VOCs (q-values < 0.05), composed of aldehydes, alcohols, nitrogen-containing compounds, hydrocarbons, ketones, aromatic compounds, esters, ethers, and carboxylic acid. From this group of volatiles, we identify candidate biomarkers that distinguish S. pyogenes from other species and establish highly produced VOCs that indicate the presence of S. pyogenes in vitro, supporting future breath-based diagnostic testing for streptococcal pharyngitis. IMPORTANCE Acute pharyngitis accounts for approximately 15 million ambulatory care visits in the United States. The most common and important bacterial cause of pharyngitis is Streptococcus pyogenesis, accounting for 15%-30% of pediatric pharyngitis. Distinguishing between bacterial and viral pharyngitis is key to management in US practice. The culture of a specimen obtained by a throat swab is the standard laboratory procedure for the microbiologic confirmation of pharyngitis; however, this method is time-consuming, which delays appropriate treatment. If left untreated, S. pyogenes pharyngitis may lead to local and distant complications. In this study, we characterized the volatile metabolomes of S. pyogenes and other related oropharyngeal colonizing bacterial species. We identify candidate biomarkers that distinguish S. pyogenes from other species and provide evidence to support future breath-based diagnostic testing for streptococcal pharyngitis.

A rare case of incarcerated appendix in a port site hernia: A case report.

INTRODUCTION AND IMPORTANCE: Hernias containing the vermiform appendix are very rare. The more common of these have eponyms, such as Amyand's hernia (incidence of 0.5-1 %) and de Garengeot's hernia (incidence of 0.8-1 %). Laparoscopic port site hernias containing the vermiform appendix are even more obscure with only seven previously reported cases. PRESENTATION OF CASE: A 71-year-old male presented with a palpable, non-reducible right lateral periumbilical mass, diagnosed preoperatively as an irreducible hernia at a port site from prior laparoscopic surgery. He had previously undergone laparoscopic bilateral inguinal hernia repairs with the 10 mm right lateral periumbilical port site defect within the musculoaponeurotic abdominal wall. DISCUSSION: There are recommendations regarding the closure of most 10-12 mm port sites, and all 15 mm port sites, given most port site hernias are associated with larger trocars. It is reasonable to conclude that if our patient's 10 mm right lateral periumbilical port site received fascial closure, the resultant hernia may have been prevented. CONCLUSION: Fascial closure of port sites >5 mm poses an easy and effective way to reduce risk of port site hernias as well as other potential complications, therein reducing readmission, need for additional surgery, and improving patient quality of life.

Argininosuccinate lyase deficiency causes blood-brain barrier disruption via nitric oxide-mediated dysregulation of claudin expression.

Nitric oxide (NO) is a critical signaling molecule that has been implicated in the pathogenesis of neurocognitive diseases. Both excessive and insufficient NO production have been linked to pathology. Previously, we have shown that argininosuccinate lyase deficiency (ASLD) is a novel model system to investigate cell-autonomous, nitric oxide synthase-dependent NO deficiency. Humans with ASLD are at increased risk for developing hyperammonemia due to a block in ureagenesis. However, natural history studies have shown that individuals with ASLD have multisystem disease including neurocognitive deficits that can be independent of ammonia. Here, using ASLD as a model of NO deficiency, we investigated the effects of NO on brain endothelial cells in vitro and the blood-brain barrier (BBB) in vivo. Knockdown of ASL in human brain microvascular endothelial cells (HBMECs) led to decreased transendothelial electrical resistance, indicative of increased cell permeability. Mechanistically, treatment with an NO donor or inhibition of Claudin-1 improved barrier integrity in ASL-deficient HBMECs. Furthermore, in vivo assessment of a hypomorphic mouse model of ASLD showed increased BBB leakage, which was partially rescued by NO supplementation. Our results suggest that ASL-mediated NO synthesis is required for proper maintenance of brain microvascular endothelial cell functions as well as BBB integrity.

MEPicides: α,ß-unsaturated Fosmidomycin N-Acyl Analogs as Efficient Inhibitors of Plasmodium falciparum 1-Deoxy-d-xylulose-5-phosphate reductoisomerase.

Malaria, a mosquito-borne disease caused by several parasites of the Plasmodium genus, remains a huge threat to global public health. There are an estimated 0.5 million malaria deaths each year, mostly among African children. Unlike humans, Plasmodium parasites and a number of important pathogenic bacteria employ the methyl erythritol phosphate (MEP) pathway for isoprenoid synthesis. Thus, the MEP pathway represents a promising set of drug targets for antimalarial and antibacterial compounds. Here, we present new unsaturated MEPicide inhibitors of 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), the second enzyme of the MEP pathway. A number of these compounds have demonstrated robust inhibition of Plasmodium falciparum DXR, potent antiparasitic activity, and low cytotoxicity against HepG2 cells. Parasites treated with active compounds are rescued by isopentenyl pyrophosphate, the product of the MEP pathway. With higher levels of DXR substrate, parasites acquire resistance to active compounds. These results further confirm the on-target inhibition of DXR in parasites by the inhibitors. Stability in mouse liver microsomes is high for the phosphonate salts, but remains a challenge for the prodrugs. Taken together, the potent activity and on-target mechanism of action of this series further validate DXR as an antimalarial drug target and the α,ß-unsaturation moiety as an important structural component.

Prevalence of DDC genotypes in patients with aromatic L-amino acid decarboxylase (AADC) deficiency and in silico prediction of structural protein changes.

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare autosomal recessive genetic disorder affecting the biosynthesis of dopamine, a precursor of both norepinephrine and epinephrine, and serotonin. Diagnosis is based on the analysis of CSF or plasma metabolites, AADC activity in plasma and genetic testing for variants in the DDC gene. The exact prevalence of AADC deficiency, the number of patients, and the variant and genotype prevalence are not known. Here, we present the DDC variant (n = 143) and genotype (n = 151) prevalence of 348 patients with AADC deficiency, 121 of whom were previously not reported. In addition, we report 26 new DDC variants, classify them according to the ACMG/AMP/ACGS recommendations for pathogenicity and score them based on the predicted structural effect. The splice variant c.714+4A>T, with a founder effect in Taiwan and China, was the most common variant (allele frequency = 32.4%), and c.[714+4A>T];[714+4A>T] was the most common genotype (genotype frequency = 21.3%). Approximately 90% of genotypes had variants classified as pathogenic or likely pathogenic, while 7% had one VUS allele and 3% had two VUS alleles. Only one benign variant was reported. Homozygous and compound heterozygous genotypes were interpreted in terms of AADC protein and categorized as: i) devoid of full-length AADC, ii) bearing one type of AADC homodimeric variant or iii) producing an AADC protein population composed of two homodimeric and one heterodimeric variant. Based on structural features, a score was attributed for all homodimers, and a tentative prediction was advanced for the heterodimer. Almost all AADC protein variants were pathogenic or likely pathogenic.

Tale of the Titers: Serologic Testing for SARS-CoV-2-Yes, No, and Maybe, With Clinical Examples From the IDSA Diagnostics Committee.

Diagnosis of acute severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection relies on detection of viral antigens or amplified viral nucleic acids. Serology, although invaluable for epidemiology, is not routinely needed clinically. However, in some settings, serologic data may have direct clinical utility: for example, in evaluation of persistent symptoms in patients without a prior diagnosis of acute infection. In contrast, SARS-CoV-2 serologic testing is sometimes used or requested in situations in which existing data do not support it, such as determination of need for vaccination. In this study, we describe available methods of serologic testing and provide cases supported by clinical vignettes of where such tests can be helpful, as well as examples where they are not. These examples may help clarify clinical decision making in this rapidly evolving area.

GAPDH mediates drug resistance and metabolism in Plasmodium falciparum malaria parasites.

Efforts to control the global malaria health crisis are undermined by antimalarial resistance. Identifying mechanisms of resistance will uncover the underlying biology of the Plasmodium falciparum malaria parasites that allow evasion of our most promising therapeutics and may reveal new drug targets. We utilized fosmidomycin (FSM) as a chemical inhibitor of plastidial isoprenoid biosynthesis through the methylerythritol phosphate (MEP) pathway. We have thus identified an unusual metabolic regulation scheme in the malaria parasite through the essential glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Two parallel genetic screens converged on independent but functionally analogous resistance alleles in GAPDH. Metabolic profiling of FSM-resistant gapdh mutant parasites indicates that neither of these mutations disrupt overall glycolytic output. While FSM-resistant GAPDH variant proteins are catalytically active, they have reduced assembly into the homotetrameric state favored by wild-type GAPDH. Disrupted oligomerization of FSM-resistant GAPDH variant proteins is accompanied by altered enzymatic cooperativity and reduced susceptibility to inhibition by free heme. Together, our data identifies a new genetic biomarker of FSM-resistance and reveals the central role of GAPDH in MEP pathway control and antimalarial sensitivity.

Prevention of malaria in pregnancy: The threat of sulfadoxine-pyrimethamine resistance.

Malaria infection in pregnancy can lead to adverse outcomes for both the pregnant person and fetus. The administration of intermittent preventative therapy (IPTp) with sulfadoxine-pyrimethamine (SP) during pregnancy (IPTp-SP) improves outcomes, including severe maternal anemia, placental malaria infection, and low infant birth weight. The WHO recommends IPTp-SP for pregnant individuals living in areas of moderate or high malaria transmission in Africa. The current regimen consists of two or more doses of SP starting as early as possible in the second trimester, at least 1 month apart. Unfortunately, rising Plasmodium falciparum SP resistance throughout Africa threatens to erode the benefits of SP. Recent studies have shown a decrease in IPTp-SP efficacy in areas with high SP resistance. Thus, there is an urgent need to identify new drug regimens that can be used for intermittent preventative therapy in pregnancy. In this review, we discuss recent data on P. falciparum SP resistance in Africa, the effect of resistance on IPTp-SP, and studies of alternative IPTp regimens. Finally, we present a framework for the ideal pharmacokinetic and pharmacodynamic properties for future IPTp regimens.

Adenosine sulfamates: Next generation of antimalarials.

Tackling the ancient infectious foe of malaria, Xie et al. (2022) uncover a novel class of nucleoside analogs that selectively hijack and inhibit the tyrosine tRNA synthase of the parasite. With high potency, good oral bioavailability, and minimal host cell toxicity, these inhibitors show promise as next-generation antimalarials.