Disrupted Mitochondrial Network Drives Deficits of Learning and Memory in a Mouse Model of FOXP1 Haploinsufficiency

Reduced cognitive flexibility, characterized by restricted interests and repetitive behavior, is associated with atypical memory performance in autism spectrum disorder (ASD), suggesting hippocampal dysfunction. FOXP1 syndrome is a neurodevelopmental disorder characterized by ASD, language deficits, global developmental delay, and mild to moderate intellectual disability. Strongly reduced Foxp1 expression has been detected in the hippocampus of Foxp1+/− mice, a brain region required for learning and memory. To investigate learning and memory performance in these animals, fear conditioning tests were carried out, which showed impaired associative learning compared with wild type (WT) animals. To shed light on the underlying mechanism, we analyzed various components of the mitochondrial network in the hippocampus. Several proteins regulating mitochondrial biogenesis (e.g., Foxo1, Pgc-1α, Tfam) and dynamics (Mfn1, Opa1, Drp1 and Fis1) were significantly dysregulated, which may explain the increased mitophagy observed in the Foxp1+/− hippocampus. The reduced activity of complex I and decreased expression of Sod2 most likely increase the production of reactive oxygen species and the expression of the pre-apoptotic proteins Bcl-2 and Bax in this tissue. In conclusion, we provide evidence that a disrupted mitochondrial network and the resulting oxidative stress in the hippocampus contribute to the altered learning and cognitive impairment in Foxp1+/− mice, suggesting that similar alterations also play a major role in patients with FOXP1 syndrome.

The effects of lisdexamfetamine dimesylate on eating behaviour and homeostatic, reward and cognitive processes in women with binge-eating symptoms: an experimental medicine study

Lisdexamfetamine dimesylate (LDX) is the only drug currently approved by the FDA for the treatment of Binge-Eating Disorder (BED), but little is known about the behavioural mechanisms that underpin the efficacy of LDX in treating BED. We examined the behavioural and neural effects of an acute dose of LDX (50 mg) in 22 women with binge-eating symptomatology using a randomised, crossover, double-blind, placebo-controlled experimental medicine design. LDX reduced self-reported appetite ratings and intake of both a pasta meal and a palatable cookie snack. LDX also decreased the eating rate of pasta but not of cookies and reduced self-reported liking ratings for pasta at the end of the meal. When viewing food pictures during an fMRI scan, LDX reduced activity bilaterally in the thalamus. LDX enhanced sustained attention and reduced impulsive responding in a continuous performance task but had no effect on emotional bias or working memory. These results suggest the observed effects of LDX on food intake (and by implication the efficacy of LDX in treating BED) may be related to the actions of the drug to enhance satiety, reduce food-related reward responding when full and/or increase cognitive control. Novel pharmacotherapies for BED might be most effective if they have a broad spectrum of effects on appetite, reward and cognition.

Measurements of 5,6 orthoquinone, surrogate for presumed active primaquine metabolite 5-hydroxyprimaquine, in the urine of Cambodian adults

The active metabolites of primaquine, in particular 5-hydroxyprimaquine, likely responsible for clearance of dormant hypnozoites, are produced through the hepatic CYP450 2D6 (CYP2D6) enzymatic pathway. With the inherent instability of 5-hydroxyprimaquine, a stable surrogate, 5,6 orthoquinone, can now be detected and measured in the urine as part of primaquine pharmacokinetic studies. This study performed CYP450 2D6 genotyping and primaquine pharmacokinetic testing, to include urine 5,6 orthoquinone, in 27 healthy adult Cambodians, as a preliminary step to prepare for future clinical studies assessing primaquine efficacy for Plasmodium vivax infections. The CYP2D6 *10 reduced activity allele was found in 57% of volunteers, and the CYP2D6 genotypes were dominated by *1/*10 (33%) and *10/*10 (30%). Predicted phenotypes were evenly split between Normal Metabolizer (NM) and Intermediate Metabolizer (IM) except one volunteer with a gene duplication and unclear phenotype, classifying as either IM or NM. Median plasma PQ area under the curve (AUC) was lower in the NM group (460 hr*ng/mL) compared to the IM group (561 hr*ng/mL), although not statistically significant. Similar to what has been found in the US study, no 5,6 orthoquinone was detected in the plasma. The urine creatinine-corrected 5,6 orthoquinone AUC in the NM group was almost three times higher than in the IM group, with peak measurements (T max ) at 4 hours. Although there is variation among individuals, future studies examining the relationship between the levels of urine 5,6 orthoquinone and primaquine radical cure efficacy could result in a metabolism biomarker predictive of radical cure.

Identification of a distal RXFP1 gene enhancer with differential activity in fibrotic lung fibroblasts involving AP-1

Relaxin/insulin-like family peptide receptor 1 (RXFP1) mediates relaxin’s antifibrotic effects and has reduced expression in the lung and skin of patients with fibrotic interstitial lung disease (fILD) including idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc). This may explain the failure of relaxin-based anti-fibrotic treatments in SSc, but the regulatory mechanisms controlling RXFP1 expression remain largely unknown. This study aimed to identify regulatory elements of RXFP1 that may function differentially in fibrotic fibroblasts. We identified and evaluated a distal regulatory region of RXFP1 in lung fibroblasts using a luciferase reporter system. Using serial deletions, an enhancer upregulating pGL3-promoter activity was localized to the distal region between -584 to -242bp from the distal transcription start site (TSS). This enhancer exhibited reduced activity in IPF and SSc lung fibroblasts. Bioinformatic analysis identified two clusters of activator protein 1 (AP-1) transcription factor binding sites within the enhancer. Site-directed mutagenesis of the binding sites confirmed that only one cluster reduced activity (-358 to -353 relative to distal TSS). Co-expression of FOS in lung fibroblasts further increased enhancer activity. In vitro complex formation with a labeled probe spanning the functional AP-1 site using nuclear proteins isolated from lung fibroblasts confirmed a specific DNA/protein complex formation. Application of antibodies against JUN and FOS resulted in the complex alteration, while antibodies to JUNB and FOSL1 did not. Analysis of AP-1 binding in 5 pairs of control and IPF lung fibroblasts detected positive binding more frequently in control fibroblasts. Expression of JUN and FOS was reduced and correlated positively with RXFP1 expression in IPF lungs. In conclusion, we identified a distal enhancer of RXFP1 with differential activity in fibrotic lung fibroblasts involving AP-1 transcription factors. Our study provides insight into RXFP1 downregulation in fILD and may support efforts to reevaluate relaxin-based therapeutics alongside upregulation of RXFP1 transcription.

Comparative Analysis of anti-Shine- Dalgarno Function in Flavobacterium johnsoniae and Escherichia coli

The anti-Shine-Dalgarno (ASD) sequence of 16S rRNA is highly conserved across Bacteria, and yet usage of Shine-Dalgarno (SD) sequences in mRNA varies dramatically, depending on the lineage. Here, we compared the effects of ASD mutagenesis in Escherichia coli, a Gammaproteobacteria which commonly employs SD sequences, and Flavobacterium johnsoniae, a Bacteroidia which rarely does. In E. coli, 30S subunits carrying any single substitution at positions 1,535–1,539 confer dominant negative phenotypes, whereas subunits with mutations at positions 1,540–1,542 are sufficient to support cell growth. These data suggest that CCUCC (1,535–1,539) represents the functional core of the element in E. coli. In F. johnsoniae, deletion of three ribosomal RNA (rrn) operons slowed growth substantially, a phenotype largely rescued by a plasmid-borne copy of the rrn operon. Using this complementation system, we found that subunits with single mutations at positions 1,535–1,537 are as active as control subunits, in sharp contrast to the E. coli results. Moreover, subunits with quadruple substitution or complete replacement of the ASD retain substantial, albeit reduced, activity. Sedimentation analysis revealed that these mutant subunits are overrepresented in the subunit fractions and underrepresented in polysome fractions, suggesting some defect in 30S biogenesis and/or translation initiation. Nonetheless, our collective data indicate that the ASD plays a much smaller role in F. johnsoniae than in E. coli, consistent with SD usage in the two organisms.

Bacterial Endophytes as a Promising Approach to Enhance the Growth and Accumulation of Bioactive Metabolites of Three Species of Chenopodium Sprouts

Sprouts are regarded as an untapped source of bioactive components that display various biological properties. Endophytic bacterium inoculation can enhance plant chemical composition and improve its nutritional quality. Herein, six endophytes (Endo 1 to Endo 6) were isolated from Chenopodium plants and morphologically and biochemically identified. Then, the most active isolate Endo 2 (strain JSA11) was employed to enhance the growth and nutritive value of the sprouts of three Chenopodium species, i.e., C. ambrosoides, C. ficifolium, and C. botrys. Endo 2 (strain JSA11) induced photosynthesis and the mineral uptake, which can explain the high biomass accumulation. Endo 2 (strain JSA11) improved the nutritive values of the treated sprouts through bioactive metabolite (antioxidants, vitamins, unsaturated fatty acid, and essential amino acids) accumulation. These increases were correlated with increased amino acid levels and phenolic metabolism. Consequently, the antioxidant activity of the Endo 2 (strain JSA11)-treated Chenopodium sprouts was enhanced. Moreover, Endo 2 (strain JSA11) increased the antibacterial activity against several pathogenic bacteria and the anti-inflammatory activities as evidenced by the reduced activity of cyclooxygenase and lipoxygenase. Overall, the Endo 2 (strain JSA11) treatment is a successful technique to enhance the bioactive contents and biological properties of Chenopodium sprouts.

Ca2+ oscillations, waves, and networks in islets from human donors with and without type 2 diabetes

Pancreatic islets are highly interconnected structures that produce pulses of insulin and other hormones, maintaining normal homeostasis of glucose and other nutrients. Normal stimulus-secretion and intercellular coupling are essential to regulated secretory responses and these hallmarks are known to be altered in diabetes. In the present study, we used calcium imaging of isolated human islets to assess their collective cell behavior. The activity occurred in the form of calcium oscillations, was synchronized across different regions of islets through calcium waves, and was glucose-dependent: higher glucose enhanced the activity, elicited a greater proportion of global calcium waves, and led to denser and less fragmented functional networks. Hub regions were identified in stimulatory conditions, and they represented the most active islet regions. Moreover, calcium waves were found to be initiated in different subregions and the roles of initiators and hubs did not overlap. In type 2 diabetes, glucose-dependence was retained, but a reduced activity, locally restricted waves, and more segregated networks were detected compared with control islets. Interestingly, hub regions seemed to suffer the most by losing a disproportionately large fraction of connections. These changes affected islets from donors with diabetes in a heterogeneous manner.

Fifteen-minute update: International normalised ratio as the treatment end point in children with acute paracetamol poisoning

Paracetamol is one of the most frequent reasons for poisonings across the UK with an estimated 90,000 patients and 150 deaths annually. International normalised ratio (INR) may be elevated due to hepatocellular damage and is frequently used to monitor progress on N-acetyl cysteine. N-acetyl cysteine is associated with reduced activity of vitamin K dependent clotting factors leading to a benign elevation of INR. In asymptomatic children with normal aspartate transaminase/alanine transaminase, isolated borderline elevation of INR following paracetamol overdose should be reviewed for possible N-acetyl cysteine induced elevation of INR. Due to these factors, in those with borderline persistent elevation of INR, N-acetyl cysteine can be safety stopped if INR is falling on two or more consecutive tests and is <3.0.

Caught in motion: human NTHL1 undergoes interdomain rearrangement necessary for catalysis

Base excision repair (BER) is the main pathway protecting cells from the continuous damage to DNA inflicted by reactive oxygen species. BER is initiated by DNA glycosylases, each of which repairs a particular class of base damage. NTHL1, a bifunctional DNA glycosylase, possesses both glycolytic and β-lytic activities with a preference for oxidized pyrimidine substrates. Defects in human NTHL1 drive a class of polyposis colorectal cancer. We report the first X-ray crystal structure of hNTHL1, revealing an open conformation not previously observed in the bacterial orthologs. In this conformation, the six-helical barrel domain comprising the helix-hairpin-helix (HhH) DNA binding motif is tipped away from the iron sulphur cluster-containing domain, requiring a conformational change to assemble a catalytic site upon DNA binding. We found that the flexibility of hNTHL1 and its ability to adopt an open configuration can be attributed to an interdomain linker. Swapping the human linker sequence for that of Escherichia coli yielded a protein chimera that crystallized in a closed conformation and had a reduced activity on lesion-containing DNA. This large scale interdomain rearrangement during catalysis is unprecedented for a HhH superfamily DNA glycosylase and provides important insight into the molecular mechanism of hNTHL1.

Hypophosphatasia

Hypophosphatasia (HPP) is an inherited metabolic disease caused by loss-of-function mutations in the tissue non-specific alkaline phosphatase (TNAP) gene. Reduced activity of TNAP leads to the accumulation of its substrates, mainly inorganic pyrophosphate and pyridoxal-5΄-phosphate, metabolic aberrations that largely explain the musculoskeletal and systemic features of the disease. More than 400 ALPL mutations, mostly missense, are reported to date, transmitted by either autosomal dominant or recessive mode. Severe disease is rare, with incidence ranging from 1:100,000 to 1:300,000 live births, while the estimated prevalence of the less severe adult form is estimated to be between 1:3100 to 1:508, in different countries in Europe. Presentation largely varies, ranging from death in utero to asymptomatic adults. In infants and children, clinical features include skeletal, respiratory and neurologic complications, while recurrent, poorly healing fractures, muscle weakness and arthropathy are common in adults. Persistently low serum alkaline phosphatase is the cardinal biochemical feature of the disease. Management requires a dedicated multidisciplinary team. In mild cases, treatment is usually symptomatic. Severe cases, with life-threating or debilitating complications, can be successfully treated with enzyme replacement therapy with asfotase alfa.