Autism Spectrum Disorder in Two Unrelated Patients with Homozygous Variants in Either ALG8 or ALG11.

Background: Autism spectrum disorder (ASD) is used to describe individuals with a specific combination of disorders in social communication and repetitive behaviors, highly restricted interests, and/or sensory behavior that begin early in life. The prevalence of ASD has been increasing rapidly in recent years. Pathophysiology of ASDs remains still unclear; however, genetic defects and multifactorial causes have been reported to play an important role in genetic disorders. The prevalence of inborn errors of metabolism (IEM) reported among patients with ASD is 2-5%. The clinical presentation of congenital disorders of glycosylation (CDG) may be in the form of psychiatric disorder only. Case Study: Case 1: a 5-year-old female patient was admitted for investigation of ASD. She had a dysmorphic facial appearance, inverted nipples, abnormal fat distribution, ataxic gait, and autistic features. Her transferrin isoelectric focusing test was compatible with a type 1 CDG pattern. A homozygous variant in ALG8 gene revealed the diagnosis of ALG8-CDG (CDG Type 1H). Case 2: a 2-year-old male patient was admitted with complaints of ASD for investigation of an underlying IEM due to speech delay. Physical examination revealed hypertelorism, small hands, and autistic behavior. Transferrin isoelectric focusing test was also found normal. As a result of the WES, a homozygous variant was detected in ALG11 confirming the diagnosis of CDG type 1p. Conclusion: CDG should also be considered in the differential diagnosis of autistic patients with dysmorphic findings. The aim of our study was to emphasize that autism should be listed among the neurological findings of CDG.

Combination of metformin/efavirenz/fluoxetine exhibits profound anticancer activity via a cancer cell-specific ROS amplification.

The possible anticancer activity of combination (M + E + F) of metformin (M), efavirenz (E), and fluoxetine (F) was investigated in normal HDF cells and HCT116 human colon cancer cells. Metformin increased cellular FOXO3a, p-FOXO3a, AMPK, p-AMPK, and MnSOD levels in HDFs but not in HCT116 cells. Cellular ATP level was decreased only in HDFs by metformin. Metformin increased ROS level only in HCT116 cells. Transfection of si-FOXO3a into HCT116 reversed the metformin-induced cellular ROS induction, indicating that FOXO3a/MnSOD is the key regulator for cellular ROS level. Viability readout with M, E, and F alone decreased slightly, but the combination of three drugs dramatically decreased cell survival in HCT116, A549, and SK-Hep-1 cancer cells but not in HDF cells. ROS levels in HCT116 cells were massively increased by M + E + F combination, but not in HDF cells. Cell cycle analysis showed that of M + E + F combination caused cell death only in HCT116 cells. The combination of M + E + F reduced synergistically mitochondrial membrane potential and mitochondrial electron transport chain complex I and III activities in HCT116 cells when compared with individual treatments. Western blot analysis indicated that DNA damage, apoptosis, autophagy, and necroptosis-realated factors increased in M + E + F-treated HCT116 cells. Oral administration with M + E + F combination for 3 weeks caused dramatic reductions in tumor volume and weight in HCT116 xenograft model of nude mice when compared with untreated ones. Our results suggest that M + E + F have profound anticancer activity both in vitro and in vivo via a cancer cell-specific ROS amplification (CASRA) through ROS-induced DNA damage, apoptosis, autophagy, and necroptosis.

Safety of COVID-19 vaccines in children with inborn errors of metabolism in terms of developing metabolic decompensation.

AIM: There are no recommended guidelines or clinical studies on safety of COVID-19 vaccines in patients with inborn errors of metabolism (IEMs). Here, we aimed to examine the relationship between COVID-19 vaccination and metabolic outcome in paediatric IEM patients. METHODS: Patients with IEM between the ages of 12 and 18 were enrolled. Term metabolic decompensation was defined as acute disruption in metabolic homeostasis due to vaccination. Clinical and biochemical markers were compared between pre- and post-vaccination periods. RESULTS: Data from a total of 36 vaccination episodes in 18 patients were included. Thirteen patients had intoxication-type metabolic disorders including organic acidemia (OA), urea cycle disorders (UCDs), maple syrup urine disease (MSUD) and phenylketonuria (PKU); 4 patients had energy metabolism disorders including fatty acid metabolism disorders and LIPIN 1 deficiency; and 1 patient had glycogen storage disorder (GSD) type 5. Seventeen patients received BNT162b2, and 1 received CoronaVac because of an underlying long QT syndrome. Fatty acid metabolism disorders, LIPIN 1 deficiency and GSD type 5 were included in the same group named 'metabolic myopathies'. In two PKU patients, plasma phenylalanine level increased significantly within 24 h following the second dose of vaccination. None of the OA, UCD, MSUD and metabolic myopathy patients experienced acute metabolic attack and had emergency department admission due to metabolic decompensation within 1 month after vaccination. CONCLUSIONS: COVID-19 vaccines did not cause acute metabolic decompensation in a cohort of 18 children with IEM.

FOXO3a Mediates Homologous Recombination Repair (HRR) via Transcriptional Activation of MRE11, BRCA1, BRIP1, and RAD50.

To test whether homologous recombination repair (HRR) depends on FOXO3a, a cellular aging model of human dermal fibroblast (HDF) and tet-on flag-h-FOXO3a transgenic mice were studied. HDF cells transfected with over-expression of wt-h-FOXO3a increased the protein levels of MRE11, BRCA1, BRIP1, and RAD50, while knock-down with siFOXO3a decreased them. The protein levels of MRE11, BRCA1, BRIP1, RAD50, and RAD51 decreased during cellular aging. Chromatin immunoprecipitation (ChIP) assay was performed on FOXO3a binding accessibility to FOXO consensus sites in human MRE11, BRCA1, BRIP1, and RAD50 promoters; the results showed FOXO3a binding decreased during cellular aging. When the tet-on flag-h-FOXO3a mice were administered doxycycline orally, the protein and mRNA levels of flag-h-FOXO3a, MRE11, BRCA1, BRIP1, and RAD50 increased in a doxycycline-dose-dependent manner. In vitro HRR assays were performed by transfection with an HR vector and I-SceI vector. The mRNA levels of the recombined GFP increased after doxycycline treatment in MEF but not in wt-MEF, and increased in young HDF comparing to old HDF, indicating that FOXO3a activates HRR. Overall, these results demonstrate that MRE11, BRCA1, BRIP1, and RAD50 are transcriptional target genes for FOXO3a, and HRR activity is increased via transcriptional activation of MRE11, BRCA1, BRIP1, and RAD50 by FOXO3a.

BNT162b2 COVID-19 vaccination elicited protective robust immune responses in pediatric patients with inborn errors of metabolism.

Introduction: SARS-CoV-2 infection can lead to a life-threatening acute metabolic decompensation in children with inborn errors of metabolism (IEM), so vaccination is mandatory. However, IEMs can also impair innate or adaptive immunity, and the impact of these immune system alterations on immunogenicity and vaccine efficacy is still unknown. Here, we investigated humoral immune responses to the BNT162b2 mRNA COVID-19 vaccine and clinical outcomes in pediatric IEM patients. Methods: Fifteen patients between 12-18 years of age with a confirmed diagnosis of IEM, and received BNT162b2 were enrolled to the study. Patients with an anti-SARS-CoV-2 IgG concentration >50 AU/mL before vaccination were defined as "COVID-19 recovered" whereas patients with undetectable anti-SARS-CoV-2 IgG concentration were defined as "COVID-19 naïve". Anti-SARS-CoV-2 Immunoglobulin G (IgG) and SARS-CoV-2 neutralizing antibody (nAb) titers were measured to assess humoral immune response. Results: Anti-SARS-CoV-2 IgG titers and nAb IH% increased significantly after the first dose. The increase in antibody titers after first and second vaccination remained significant in COVID-19 naïve patients. Complete anti-SARS-CoV-2 IgG seropositivity and nAb IH% positivity was observed in all patients after the second dose. Vaccination appears to be clinically effective in IEM patients, as none of the patients had COVID-19 infection within six months of the last vaccination. Discussion: Humoral immune response after two doses of BNT162b2 in pediatric IEM patients was adequate and the immune response was not different from that of healthy individuals.

Inborn errors of metabolism and coronavirus disease 2019: Evaluation of the metabolic outcome.

BACKGROUND: Infectious diseases can result in a catabolic state and possibly trigger an acute metabolic decompensation in inborn errors of metabolism (IEM), which could be life threatening. Studies regarding the course of severe acute respiratory syndrome coronavirus 2 infections in patients with IEM are generally limited to case reports. Here, we aimed to evaluate the clinical findings of coronavirus disease 2019 (COVID-19) and describe the impact of severe acute respiratory syndrome coronavirus 2 infections on metabolic outcomes in IEM patients. METHODS: Patients who were diagnosed with different types of IEM and developed microbiologically confirmed COVID-19 infection were included. Clinical findings and laboratory results were recorded retrospectively in terms of both IEM and COVID-19. RESULTS: Eleven patients with diagnosis of intoxication type metabolic disorders, five patients with energy metabolism disorders, and six patients with complex molecular disorders were enrolled. The most frequent clinical finding was fever (52.1%) followed by fatigue/myalgia (47.8%). None of the patients was younger than 1 year. None of the patients presented severe or critical disease. In terms of metabolic decompensation, two patients diagnosed with propionic acidemia, one patient with methylmalonic acidemia and one patient with 3-hydroxy-3-methylglutaryl-CoA lyase deficiency presented clinical and biochemical findings of an acute metabolic attack. CONCLUSIONS: Based on our results, IEM are not found to be an additional risk factor for severe COVID-19 infection. However, patients with intoxication type and energy metabolism disorders should be considered as a vulnerable population for COVID-19 and have a major risk of developing acute metabolic decompensation that can lead to life-threatening complications.

Metformin-induced ROS upregulation as amplified by apigenin causes profound anticancer activity while sparing normal cells.

Metformin increased cellular ROS levels in AsPC-1 pancreatic cancer cells, with minimal effect in HDF, human primary dermal fibroblasts. Metformin reduced cellular ATP levels in HDF, but not in AsPC-1 cells. Metformin increased AMPK, p-AMPK (Thr172), FOXO3a, p-FOXO3a (Ser413), and MnSOD levels in HDF, but not in AsPC-1 cells. p-AMPK and p-FOXO3a also translocated from the cytosol to the nucleus by metformin in HDF, but not in AsPC-1 cells. Transfection of si-FOXO3a in HDF increased ROS levels, while wt-FOXO3a-transfected AsPC-1 cells decreased ROS levels. Metformin combined with apigenin increased ROS levels dramatically and decreased cell viability in various cancer cells including AsPC-1 cells, with each drug used singly having a minimal effect. Metformin/apigenin combination synergistically decreased mitochondrial membrane potential in AsPC-1 cells but to a lesser extent in HDF cells. Metformin/apigenin combination in AsPC-1 cells increased DNA damage-, apoptosis-, autophagy- and necroptosis-related factors, but not in HDF cells. Oral administration with metformin/apigenin caused dramatic blocks tumor size in AsPC-1-xenografted nude mice. Our results suggest that metformin in cancer cells differentially regulates cellular ROS levels via AMPK-FOXO3a-MnSOD pathway and combination of metformin/apigenin exerts anticancer activity through DNA damage-induced apoptosis, autophagy and necroptosis by cancer cell-specific ROS amplification.

Purple corn extract (PCE) alleviates cigarette smoke (CS)-induced DNA damage in rodent blood cells by activation of AMPK/Foxo3a/MnSOD pathway.

Purple corn extract (PCE) is a nutraceutical, an activator of AMPK, and it has antioxidants and anticancer properties. Therefore, PCE could be a candidate for alleviating cigarette smoke (CS)-induced oxidative DNA damage. This study examined whether PCE can have a protective effect on blood cells in an animal model of cigarette smoke (CS)-induced DNA damage. PCE was orally administered to CS-inhaled Spraque-Dawley (SD) rats, followed by the target cells being examined for markers of DNA damage. The study also sought to elucidate the mechanism of PCE action in the PCE treated animals. SD rat inhalation of CS was for once a day for 30 min, repeated for 7 days. PCE was administered orally before CS inhalation. Pretreatment of the animals with oral PCE kept the numbers of white blood cells (WBC) as well as neutrophils (NE), lymphocytes (LY), monocytes (Mo), eosinophils (EO), abd jasophils (BA) from increasing as those were increased in the CS-inhaling SD rats. The amount of phosphorylated γ-H2AX, a DNA damage marker, was assayed in the circulating blood cells collected from the animals and western blot analysis with anti-Foxo3a, p-Foxo3a, p-AMPK, MnSOD antibodies were performed on those cells. PCE protected the circulating blood cells from CS inhalation-induced DNA damage by 44% as assayed by increases in γ-H2AX. PCE also increased the nuclear localization of Foxo3a by 52% over control cells. Mechanistically, PCE appears to efficiently protect various blood cell types from CS-induced DNA damage through removal of ROS via activation of the AMPK/Foxo3a/MnSOD pathway.

Synergistic Activity of Ceragenins Against Carbapenem-Resistant Acinetobacter baumannii Strains in Both Checkerboard and Dynamic Time-Kill Assays.

Acinetobacter baumannii is an emerging opportunistic pathogen that primarily infects critically ill patients in nosocomial settings and there is a need for identifying new alternative therapeutic agents against these organisms. Ceragenins are non-peptide, membrane-active agents that mimic the antimicrobial properties of antimicrobial peptides (AMPs) and affect the membrane permeability of microorganisms. The in vitro activities of CSA-8, CSA-13, CSA-44, CSA-131, CSA-138 either alone or in combination with colistin (sulphate) were determined against 25 carbapenem-resistant A. baumannii strains. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of selected ceragenins and colistin against these isolates were measured by in vitro microbroth dilution techniques. Checkerboard techniques and time-kill assays were performed to determine the activities of combinations. The MIC50 values (mg/L) of CSA-8, CSA-13, CSA-44, CSA-131, CSA-138 and colistin were 32, 4, 8, 2, 4 and 0.5, respectively. The MIC90 (mg/L) of CSA-8, CSA-13, CSA-44, CSA-131, CSA-138 and colistin were 128, 8, 16, 8, 16 and 16, respectively. At 6 h, 1×MIC and 2×MIC of CSA-13 were bactericidal. CSA-13 + colistin combination displayed synergistic interaction. Antagonism between antimicrobials was not observed. According to the results, CSA-13 and CSA-131 can be good alternatives for infections caused by carbapenem-resistant A. baumannii.

Antibacterial and Antibiofilm Activities of Ceragenins against Pseudomonas aeruginosa Clinical Isolates.

OBJECTIVES: Pseudomonas aeruginosa can cause life-threatening infections that are difficult to treat due to its high resistance to antibiotics and its ability to form antibiotic tolerant biofilms. Ceragenins, designed to mimic the activities of antimicrobial peptides, represent a promising new group of antibacterial agents that display potent anti-P. aeruginosa activity. The aim of this study was to evaluate the antibacterial and antibiofilm activities of ceragenins in comparison to colistin and ciprofloxacin against P. aeruginosa strains. MATERIALS AND METHODS: Biofilm formation and determination of minimum inhibitory concentration (MIC) values of ceragenins (CSA-13, CSA-44, CSA-131, and CSA-138), ciprofloxacin, and colistin were evaluated against 25 P. aeruginosa isolates. Four good biofilm-producing strains were chosen for biofilm studies, and sessile MICs and inhibition of molecule adhesion and biofilm formation were evaluated. RESULTS: The MIC50 (µg/mL) values of CSA-13, CSA-44, CSA-131, CSA-138, ciprofloxacin, and colistin were 8, 8, 8, 16, 1, and 2, respectively. The sessile MICs for molecules were greater than planktonic MICs. CSA-13, CSA-44, and CSA-131 were more efficient after 4 h incubation while CSA-138, ciprofloxacin and colistin were more efficient after 1 h incubation. The most efficient agent for inhibition of adhesion was colistin (up to 45%). CSA-131, CSA-138, and colistin were the most efficient agents for inhibition of biofilm formation (up to 90%). CONCLUSION: Our study highlights the potential of CSA-131 and CSA-138 as potential alternative agents to conventional antibiotics for the eradication of biofilms of P. aeruginosa.

Exposure to environmental tobacco smoke by healthy children aged below five (Preliminary study).

AIM: Children are commonly affected by environmental tobacco smoke. The presence of exposure can be deduced from urinary urine kotinine/creatinine ratio and history. The aim of this study was to investigate passive smoking in healthy children between one-month and five year old, and to determine the adverse effects of passive smoking on child health. MATERIAL AND METHODS: Children between one-month and five year old who were regularly monitored for health were included following informed consent given by their parents. The questionnaire method was used. Demographic variables, respiratory tract infections, recurrent infections were questioned. The levels of cotinine, creatinine were measured and the cotinine/creatinine ratios were calculated in urine specimens taken from the children. Growth status and infection frequency were determined using demographic data, cotinine/creatinine ratios in urine, exposure rate to second-hand tobacco smoke of the children. RESULTS: The ratio of household smokers was 70.3%, the ratio of non-smokers was 29.7%. Fifty percent of the mothers were smokers. Urinary cotinine/creatinine ratios were found to be significantly higher in children of smokers compared with children of non-smokers (p=0.011). One third of the children was evaluated as passive smokers. The presence of a smoker at home and the increase in the number of cigarettes smoked during the day increased the frequency of acute respiratory infections (p=0.047). CONCLUSION: In these regularly-monitored preschool children, we found frequent exposure to cigarette smoke. This study contributes to national data and will aid in increasing the awareness for the deleterious effects of passive smoking on child health.