Chiral Memory in Dynamic Transformation from Porous Organic Cages to Covalent Organic Frameworks for Enantiorecognition Analysis.

The preservation of chirality during a transformation process, known as the "chiral memory" effect, has garnered significant attention across multiple research disciplines. Here, we first report the retention of the original chiral structure during dynamic covalent chemistry (DCC)-induced structural transformation from porous organic cages into covalent organic frameworks (COFs). A total of six two-dimensional chiral COFs constructed by entirely achiral building blocks were obtained through the DCC-induced substitution of chiral linkers in a homochiral cage (CC3-R or -S) using achiral amine monomers. Homochirality of these COFs resulted from the construction of 3-fold-symmetric benzene-1,3,5-methanimine cores with a propeller-like configuration of one single-handedness throughout the cage-to-COF transformation. The obtained chiral COFs can be further utilized as fluorescence sensors or chiral stationary phases for gas chromatography with high enantioselectivity. The present study thus highlighted the great potential to expand the scope of functional chiral materials via DCC-induced crystal-to-crystal transformation with the chiral memory effect.

[Preparation of porous organic cage and its use as chiral stationary phase for capillary electrochromatography].

Porous organic cages (POCs) are a unique type of microporous materials composed of discrete molecules with internal cavities that are accessible to various compounds. In this study, a prismatic chiral POC with good thermochemical stability was synthesized by condensing (1R,2R)-diaminocyclohexane and 3,3',5,5'-teturonic-4,4'-biphenediol via the Schiff base reaction and characterized by proton nuclear magnetic resonance spectroscopy, infrared (IR) spectroscopy, thermogravimetric analysis (TGA), and scanning electron microscopy. The IR spectrum of the POC revealed a strong characteristic absorption peak at 1635 cm-1, indicating that it formed imine bonds (C=N). The absorption peak at 3425 cm-1 was attributed to the stretching vibrations of -OH, the absorption peaks at 2925 and 2858 cm-1 were attributed to the stretching vibrations of N=C-H and C-H, and the absorption peaks at 1446 and 1383 cm-1 were attributed to the stretching vibrations of C=C-H and C=C in the benzene ring. High-resolution mass spectral analysis of the POC showed a molecular ion peak at m/z 1363.7228, indicating its successful synthesis. TGA was performed from 25 to 800 ℃ at a rate of 10 ℃/min, and the results of this analysis showed that the POC was stable up to approximately 300 ℃. The POC was dissolved in dichloromethane and uniformly coated on the inner wall of a quartz capillary via the dynamic coating method to prepare a capillary electrochromatographic column. The experimental results revealed that the chiral electrochromatographic column could not only resolve ofloxacin, Troger's base, 2-amino-1-butanol, and 1-phenyl-1-amyl alcohol but also separate the isomers of o-, m-, and p-toluidine and o-, m-, and p-chloroaniline, indicating its good chiral separation ability. Investigation of the optimal separation conditions for ofloxacin, Troger's base, 2-amino-1-butanol, and 1-phenyl-1-amyl alcohol revealed that the voltage, buffer solution concentration, and pH significantly affected their separation degree. In particular, the optimal separation voltage for ofloxacin, Troger's base, and 2-amino-1-butanol was 15 kV, while that for 1-phenyl-1-amyl alcohol was 17 kV. The optimal buffer concentration and pH for ofloxacin, Troger's base, 2-amino-1-butanol, and 1-phenyl-1-amyl alcohol were 0.100 mol/L and 7.5. Under optimal chromatographic conditions, the resolution values for ofloxacin, Troger's base, 2-amino-1-butanol, and 1-phenyl-1-pentanol were 1.80, 3.33, 1.69, and 1.18, respectively. The results collectively demonstrate that the prepared POC may serve as a good chiral stationary phase for capillary electrochromatography with a certain chiral resolution ability and has good application prospects in chromatographic separation.

Zfp281 (ZBP-99) plays a functionally redundant role with Zfp148 (ZBP-89) during erythroid development.

Erythroid maturation requires the concerted action of a core set of transcription factors. We previously identified the Krüppel-type zinc finger transcription factor Zfp148 (also called ZBP-89) as an interacting partner of the master erythroid transcription factor GATA1. Here we report the conditional knockout of Zfp148 in mice. Global loss of Zfp148 results in perinatal lethality from nonhematologic causes. Selective Zfp148 loss within the hematopoietic system results in a mild microcytic and hypochromic anemia, mildly impaired erythroid maturation, and delayed recovery from phenylhydrazine-induced hemolysis. Based on the mild erythroid phenotype of these mice compared with GATA1-deficient mice, we hypothesized that additional factor(s) may complement Zfp148 function during erythropoiesis. We show that Zfp281 (also called ZBP-99), another member of the Zfp148 transcription factor family, is highly expressed in murine and human erythroid cells. Zfp281 knockdown by itself results in partial erythroid defects. However, combined deficiency of Zfp148 and Zfp281 causes a marked erythroid maturation block. Zfp281 physically associates with GATA1, occupies many common chromatin sites with GATA1 and Zfp148, and regulates a common set of genes required for erythroid cell differentiation. These findings uncover a previously unknown role for Zfp281 in erythroid development and suggest that it functionally overlaps with that of Zfp148 during erythropoiesis.

Determination of colistin in animal tissues, egg, milk, and feed by ultra-high performance liquid chromatography-tandem mass spectrometry.

A confirmatory method for the determination of colistin in animal tissues, egg, milk, and feed was developed and validated. Colistin A and colistin B were extracted from samples with the mixture of 10% trichloroacetic acid-acetonitrile and isolated with mixed-mode weak cation exchange cartridge. Analytes were separated from matrix components using ultra-high performance liquid chromatography, and detected with electrospray ionization on a triple quadrupole mass spectrometer. Mean recoveries ranged from 78.0% to 115.6% with intra-day and inter-day relative standard deviation lower than 8.4% and 12.4%, respectively. The quantitation limits for different matrices were between 5 and 30 µg/kg, which was satisfactory for surveillance monitoring. The developed method was applied to the analysis of real samples collected from different provinces of China, and 19 out of 348 samples were found to be contaminated, with the highest concentration of approximately 12,000 µg/kg colistin A and 10,000 µg/kg colistin B in feed.

Somatic mitochondrial DNA mutations do not increase neuronal vulnerability to MPTP in young POLG mutator mice.

Mitochondrial DNA (mtDNA) mutations are hypothesized to play a pathogenic role in aging and age-related neurodegenerative diseases such as Parkinson's disease (PD). In support of this, high levels of somatic mtDNA mutations in "POLG mutator" mice carrying a proofreading-deficient form of mtDNA polymerase ã (Polg(D257A)) lead to a premature aging phenotype. However, the relevance of this finding to the normal aging process has been questioned as the number of mutations is greater even in young POLG mutator mice, which shows no overt phenotype, than levels achieved during normal aging in mice. Vulnerability of dopaminergic neurons to 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) increases with age, and we hypothesized that this may result in part from the accumulation with age of somatic mtDNA mutations. If correct, then levels of mutations in young (2­3 month old) POLG mutator mice should be sufficient to increase vulnerability to MPTP. In contrast, we find that susceptibility to MPTP in both heterozygous and homozygous POLG mutator mice at this young age is not different from that of wild type littermate controls as measured by levels of tyrosine hydroxylase positive (TH+) striatal terminals, striatal dopamine and its metabolites, a marker of oxidative damage, or stereological counts of TH+ and total substantia nigra neurons. These unexpected results do not support the hypothesis that somatic mtDNA mutations contribute to the age-related vulnerability of dopaminergic neurons to MPTP. It remains possible that somatic mtDNA mutations influence vulnerability to other stressors, or require additional time for the deleterious consequences to manifest. Furthermore, the impact of the higher levels of mutations present at older ages in these mice was not assessed in our study, although a prior study also failed to detect an increase in vulnerability to MPTP in older mice. With these caveats, the current data do not provide evidence for a role of somatic mtDNA mutations in determining the vulnerability to MPTP.

Rapamycin drives selection against a pathogenic heteroplasmic mitochondrial DNA mutation.

Mitochondrial DNA (mtDNA) mutations cause a variety of mitochondrial disorders for which effective treatments are lacking. Emerging data indicate that selective mitochondrial degradation through autophagy (mitophagy) plays a critical role in mitochondrial quality control. Inhibition of mammalian target of rapamycin (mTOR) kinase activity can activate mitophagy. To test the hypothesis that enhancing mitophagy would drive selection against dysfunctional mitochondria harboring higher levels of mutations, thereby decreasing mutation levels over time, we examined the impact of rapamycin on mutation levels in a human cytoplasmic hybrid (cybrid) cell line expressing a heteroplasmic mtDNA G11778A mutation, the most common cause of Leber's hereditary optic neuropathy. Inhibition of mTORC1/S6 kinase signaling by rapamycin induced colocalization of mitochondria with autophagosomes, and resulted in a striking progressive decrease in levels of the G11778A mutation and partial restoration of ATP levels. Rapamycin-induced upregulation of mitophagy was confirmed by electron microscopic evidence of increased autophagic vacuoles containing mitochondria-like organelles. The decreased mutational burden was not due to rapamycin-induced cell death or mtDNA depletion, as there was no significant difference in cytotoxicity/apoptosis or mtDNA copy number between rapamycin and vehicle-treated cells. These data demonstrate the potential for pharmacological inhibition of mTOR kinase activity to activate mitophagy as a strategy to drive selection against a heteroplasmic mtDNA G11778A mutation and raise the exciting possibility that rapamycin may have therapeutic potential for the treatment of mitochondrial disorders associated with heteroplasmic mtDNA mutations, although further studies are needed to determine if a similar strategy will be effective for other mutations and other cell types.

Behavioral and metabolic characterization of heterozygous and homozygous POLG mutator mice.

The mitochondrial DNA (mtDNA) polymerase γ (POLG) mutator mice provide the first experimental evidence that high levels of somatic mtDNA mutations can be functionally significant. Here we report that older homozygous, but not heterozygous, POLG mice show significant reductions in striatal dopaminergic terminals as well as deficits in motor function. However, resting oxygen consumption, heat production, mtDNA content and mitochondrial electron transport chain activities are significantly decreased at older ages in both homozygous and heterozygous mice. These results indicate that high levels of somatic mtDNA mutations can contribute to dopaminergic dysfunction and to behavioral and metabolic deficits.

Pgc-1α overexpression downregulates Pitx3 and increases susceptibility to MPTP toxicity associated with decreased Bdnf.

Multiple mechanisms likely contribute to neuronal death in Parkinson's disease (PD), including mitochondrial dysfunction and oxidative stress. Peroxisome proliferator-activated receptor gamma co-activator-1 alpha (PGC-1α) positively regulates the expression of genes required for mitochondrial biogenesis and the cell's antioxidant responses. Also, expression of PGC-1α-regulated genes is low in substantia nigra (SN) neurons in early PD. Thus upregulation of PGC-1α is a candidate neuroprotective strategy in PD. Here, an adeno-associated virus (AAV) was used to induce unilateral overexpression of Pgc-1α, or a control gene, in the SN of wild-type C57BL/6CR mice. Three weeks after AAV administration, mice were treated with saline or MPTP. Overexpression of Pgc-1α in the SN induced expression of target genes, but unexpectedly it also greatly reduced the expression of tyrosine hydroxylase (Th) and other markers of the dopaminergic phenotype with resultant severe loss of striatal dopamine. Reduced Th expression was associated with loss of Pitx3, a transcription factor that is critical for the development and maintenance of dopaminergic cells. Expression of the neurotrophic factor Bdnf, which also is regulated by Pitx3, similarly was reduced. Overexpression of Pgc-1α also led to increased sensitivity to MPTP-induced death of Th+ neurons. Pgc-1α overexpression alone, in the absence of MPTP treatment, did not lead to cell loss in the SN or to loss of dopaminergic terminals. These data demonstrate that overexpression of Pgc-1α results in dopamine depletion associated with lower levels of Pitx3 and enhances susceptibility to MPTP. These data may have ramifications for neuroprotective strategies targeting overexpression of PGC-1α in PD.

Somatic mitochondrial DNA mutations in early Parkinson and incidental Lewy body disease.

Somatic mutations in mitochondrial DNA (mtDNA) are hypothesized to play a role in Parkinson disease (PD), but large increases in mtDNA mutations have not previously been found in PD, potentially because neurons with high mutation levels degenerate and thus are absent in late stage tissue. To address this issue, we studied early stage PD cases and cases of incidental Lewy body disease (ILBD), which is thought to represent presymptomatic PD. We show for the first time that mtDNA mutation levels in substantia nigra neurons are significantly elevated in this group of early PD and ILBD cases.

Association of PGC-1alpha polymorphisms with age of onset and risk of Parkinson's disease.

BACKGROUND: Peroxisome proliferator-activated receptor-γ co-activator (PGC)-1α is a transcriptional co-activator of antioxidant genes and a master regulator of mitochondrial biogenesis. Parkinson's disease (PD) is associated with oxidative stress and mitochondrial dysfunction and recent work suggests a role for PGC-1α. We hypothesized that the rs8192678 PGC-1α single nucleotide polymorphism (SNP) may influence risk or age of onset of PD. The A10398G mitochondrial SNP has been inversely associated with risk of PD in some studies. In the current study we analyzed whether rs8192678 or other PGC-1α SNPs affect PD risk or age of onset, singularly or in association with the A10398G SNP. METHODS: Genomic DNA samples from 378 PD patients and 173 age-matched controls were analyzed by multiplexed probe sequencing, followed by statistical analyses of the association of each SNP, alone or in combination, with risk or age of onset of PD. Adjustments were made for age of onset being less than the age of sampling, and for the observed dependence between these two ages. The PD samples were obtained as two separate cohorts, therefore statistical methods accounted for different sampling methods between the two cohorts, and data were analyzed using Cox regression adjusted for sampling in the risk set definition and in the model. RESULTS: The rs8192678 PGC-1α SNP was not associated with the risk of PD. However, an association of the PGC-1α rs8192678 GG variant with longevity was seen in control subjects (p=0.019). Exploratory studies indicated that the CC variant of rs6821591 was associated with risk of early onset PD (p=0.029), with PD age of onset (p=0.047), and with longevity (p=0.022). The rs2970848 GG allele was associated with risk of late onset PD (p=0.027). CONCLUSIONS: These data reveal possible associations of the PGC-1α SNPs rs6821591 and rs2970848 with risk or age of onset of PD, and of the PGC-1α rs8192678 GG and the rs6821591 CC variants with longevity. If replicated in other datasets, these findings may have important implications regarding the role of PGC-1α in PD and longevity.

Oral N-acetyl-cysteine attenuates loss of dopaminergic terminals in alpha-synuclein overexpressing mice.

Levels of glutathione are lower in the substantia nigra (SN) early in Parkinson's disease (PD) and this may contribute to mitochondrial dysfunction and oxidative stress. Oxidative stress may increase the accumulation of toxic forms of alpha-synuclein (SNCA). We hypothesized that supplementation with n-acetylcysteine (NAC), a source of cysteine--the limiting amino acid in glutathione synthesis, would protect against alpha-synuclein toxicity. Transgenic mice overexpressing wild-type human alpha-synuclein drank water supplemented with NAC or control water supplemented with alanine from ages 6 weeks to 1 year. NAC increased SN levels of glutathione within 5-7 weeks of treatment; however, this increase was not sustained at 1 year. Despite the transient nature of the impact of NAC on brain glutathione, the loss of dopaminergic terminals at 1 year associated with SNCA overexpression was significantly attenuated by NAC supplementation, as measured by immunoreactivity for tyrosine hydroxylase in the striatum (p = 0.007; unpaired, two-tailed t-test), with a similar but nonsignificant trend for dopamine transporter (DAT) immunoreactivity. NAC significantly decreased the levels of human SNCA in the brains of PDGFb-SNCA transgenic mice compared to alanine treated transgenics. This was associated with a decrease in nuclear NFkappaB localization and an increase in cytoplasmic localization of NFkappaB in the NAC-treated transgenics. Overall, these results indicate that oral NAC supplementation decreases SNCA levels in brain and partially protects against loss of dopaminergic terminals associated with overexpression of alpha-synuclein in this model.

Mitochondrial complex I gene variant associated with early age at onset in spinocerebellar ataxia type 2.

BACKGROUND: A common mitochondrial complex I gene polymorphism (10398G) is reported to be inversely associated with the risk of Parkinson disease. We hypothesized that this variant might have a protective effect on the central nervous system and therefore might delay the onset of symptoms in spinocerebellar ataxia type 2 (SCA2). OBJECTIVE: To assess the association of the 10398G polymorphism with age at onset in Cuban patients with SCA2. DESIGN: Genetic association study. SETTING: Holguin, Cuba. PATIENTS: Forty-six Cuban patients with SCA2. MAIN OUTCOME MEASURES: Presence or absence of the 10398G polymorphism was determined in 46 Cuban patients with SCA2 and early or late onset of symptoms, defined as at least 2 SDs lower than or higher than the mean age at onset for patients with a similarly sized triplet repeat expansion. RESULTS: The polymorphism was present in 11 of 27 Cuban patients with SCA2 and early onset (41%) vs 2 of 19 with late onset (11%) (Fisher exact test; P = .04). CONCLUSION: Contrary to our prediction of a later onset of SCA2 in patients with the 10398G polymorphism, we find that this variant is associated with an earlier age at onset in Cuban patients with SCA2.

Suppression of reactive oxygen species and neurodegeneration by the PGC-1 transcriptional coactivators.

PPARgamma coactivator 1alpha (PGC-1alpha) is a potent stimulator of mitochondrial biogenesis and respiration. Since the mitochondrial electron transport chain is the main producer of reactive oxygen species (ROS) in most cells, we examined the effect of PGC-1alpha on the metabolism of ROS. PGC-1alpha is coinduced with several key ROS-detoxifying enzymes upon treatment of cells with an oxidative stressor; studies with RNAi or null cells indicate that PGC-1alpha is required for the induction of many ROS-detoxifying enzymes, including GPx1 and SOD2. PGC-1alpha null mice are much more sensitive to the neurodegenerative effects of MPTP and kainic acid, oxidative stressors affecting the substantia nigra and hippocampus, respectively. Increasing PGC-1alpha levels dramatically protects neural cells in culture from oxidative-stressor-mediated death. These studies reveal that PGC-1alpha is a broad and powerful regulator of ROS metabolism, providing a potential target for the therapeutic manipulation of these important endogenous toxins.

Purkinje neuron degeneration in nervous (nr) mutant mice is mediated by a metabolic pathway involving excess tissue plasminogen activator.

Purkinje neurons (PNs), the central cells in cerebellar circuitry and function, constitute a vulnerable population in many human genetic, malignant, hypoxic, and toxic diseases. In the nervous (nr) mutant mouse, the majority of PNs die in the fourth to fifth postnatal weeks, but the responsible molecules are unknown. We first disclose a remarkable increase in mRNA expression and protein concentration in the nr cerebellum of tissue plasminogen activator (tPA), a gene closely linked to the mapped but as-yet-uncloned nr locus. Evidence that excessive tPA triggers nr PN death was obtained with organotypic slice cultures expressing the nr PN phenotype, in which an inhibitor of tPA led to increased nr PN survival. An antagonist of protein kinase C, a downstream component in the tPA pathway, also increased nr PN survival. Additional downstream targets in the tPA pathway (the mitochondrial voltage-dependent anion channel, brain-derived neurotrophic factor, and neurotrophin 3) were also abnormal, in parallel with the alterations in PN mitochondrial morphology, dendritic growth, and synaptogenesis that culminate in nr PN death and motor incoordination. We thus propose a molecular pathway by which the excessive tPA in nr cerebellum mediates PN degeneration.

Somatic mitochondrial DNA mutations in single neurons and glia.

Somatic mitochondrial DNA (mtDNA) point mutations reach high levels in the brain. However, the cell types that accumulate mutations and the patterns of mutations within individual cells are not known. We have quantified somatic mtDNA mutations in 28 single neurons and in 18 single glia from post-mortem human substantia nigra of six control subjects. Both neurons and glia contain mtDNA with somatic mutations. Single neurons harbor a geometric mean (95% CI) of 200.3 (152.9-262.4) somatic mtDNA point mutations per million base pairs, compared to 133.8 (97.5-184.9) for single glia (p=0.0251). If mutations detected multiple times in the same cell are counted only once, the mean mutation level per million base pairs remains elevated in single neurons (146.9; 124.0-174.2) compared to single glia (100.5; 81.5-126.5; p=0.009). Multiple distinct somatic point mutations are present in different cells from the same subject. Most of these mutations are individually present at low levels (less than 10-20% of mtDNA molecules), but with high aggregate mutation levels, particularly in neurons. These mutations may contribute to changes in brain function during normal aging and neurodegenerative disorders.

A common NURR1 polymorphism associated with Parkinson disease and diffuse Lewy body disease.

BACKGROUND: NURR1 plays a key role in mesencephalic dopaminergic neuron development and survival. A homozygous NURR1 polymorphism (a single base-pair insertion in intron 6) (NI6P) has been reported to be associated with Parkinson disease (PD). OBJECTIVE: To assess the association of the NI6P with PD and diffuse Lewy body disease. DESIGN: Case-control study. SETTING: Movement disorders clinic and tissue provided by brain banks. PATIENTS: Patients with pathologically proven PD (n = 37) or diffuse Lewy body disease (n = 35), neuropathologically normal control subjects (n = 59), those clinically diagnosed as having PD (n = 66), and spousal controls (n = 29). METHODS: Determining the frequency of heterozygotes and homozygotes for the NI6P by DNA sequencing and restriction endonuclease analyses. RESULTS: Overall, 41 (39.8%) of the 103 patients with PD were heterozygotes compared with 22 (25.0%) of the 88 controls (P =.03), with a relative risk (estimated from the odds ratio) for PD of 2.03 (95% confidence interval, 1.08-3.81) for heterozygotes vs wild type subjects. Heterozygotes were more frequent in the subgroup of patients with pathologically confirmed PD (18 [48.6%] of 37) vs controls (14 [23.7%] of 59) (P =.01), with a relative risk for PD of 2.84 (95% confidence interval, 1.17-6.88) for heterozygotes vs wild type subjects. In patients clinically diagnosed as having PD, heterozygotes were more frequent in early-onset cases (onset at < or =45 years) (10 [55.6%] of 18) compared with late-onset cases (onset at >45 years) (10 [23.8%] of 42) (P =.02) or spousal controls (8 [27.6%] of 29) (P =.06), with a relative risk for early-onset PD of 4.17 (95% confidence interval, 1.13-15.33) for heterozygotes vs subjects with 2 wild type alleles. The homozygous NI6P was not associated with PD, but was present in 6 (17.1%) of the 35 patients with diffuse Lewy body disease compared with 3 (5.1%) of the 59 controls (P =.06). CONCLUSIONS: The common heterozygous NI6P is associated with an increased risk of PD. An association of borderline significance was found for the homozygous NI6P and diffuse Lewy body disease.