Ophthalmate is a new regulator of motor functions via CaSR: Implications for movement disorders.

Dopamine's role as the principal neurotransmitter in motor functions has long been accepted. We broaden this conventional perspective by demonstrating the involvement of non-dopaminergic mechanisms. In mouse models of Parkinson's Disease (PD), we observed that L-DOPA elicited a substantial motor response even when its conversion to dopamine was blocked by inhibiting the enzyme aromatic amino acid decarboxylase (AADC). Remarkably, the motor activity response to L-DOPA in the presence of an AADC inhibitor (NSD1015) showed a delayed onset, yet greater intensity and longer duration, peaking at 7 hours, compared to when L-DOPA was administered alone. This suggests an alternative pathway or mechanism, independent of dopamine signaling, mediating the motor functions. We sought to determine the metabolites associated with the pronounced hyperactivity observed, using comprehensive metabolomics analysis. Our results revealed that the peak in motor activity induced by NSD1015/L-DOPA in PD mice is associated with a surge (20-fold) in brain levels of the tripeptide ophthalmic acid (OA, also known as ophthalmate in its anionic form). Interestingly, we found that administering ophthalmate directly to the brain rescued motor deficits in PD mice in a dose-dependent manner. We investigated the molecular mechanisms underlying ophthalmate's action and discovered, through radioligand binding and cAMP-luminescence assays, that ophthalmate binds to and activates the calcium-sensing receptor (CaSR). Additionally, our findings demonstrated that a CaSR antagonist inhibits the motor-enhancing effects of ophthalmate, further solidifying the evidence that ophthalmate modulates motor functions through the activation of the CaSR. The discovery of ophthalmate as a novel regulator of motor function presents significant potential to transform our understanding of brain mechanisms of movement control and the therapeutic management of related disorders.

Humanized dopamine D4.7 receptor male mice display risk-taking behavior and deficits of social recognition and working memory in light/dark-dependent manner.

The dopamine D4 receptor 7-repeat allele (D4.7 R) has been linked with psychiatric disorders such as attention-deficit-hyperactivity disorder, autism, and schizophrenia. However, the highly diverse study populations and often contradictory findings make it difficult to draw reliable conclusions. The D4.7 R has the potential to explain individual differences in behavior. However, there is still a great deal of ambiguity surrounding whether it is causally connected to the etiology of psychiatric disorders. Therefore, humanized D4.7 R mice, with the long third intracellular domain of the human D4.7 R, may provide a valuable tool to examine the relationship between the D4.7 R variant and specific behavioral phenotypes. We report that D4.7 R male mice carrying the humanized D4.7 R variant exhibit distinct behavioral features that are dependent on the light-dark cycle. The behavioral phenotype was characterized by a working memory deficit, delayed decision execution in the light phase, decreased stress and anxiety, and increased risk behavior in the dark phase. Further, D4.7 R mice displayed impaired social recognition memory in both the light and dark phases. These findings provide insight into the potential causal relationship between the human D4.7 R variant and specific behaviors and encourage further consideration of dopamine D4 receptor (DRD4) ligands as novel treatments for psychiatric disorders in which D4.7 R has been implicated.

Alzheimer's disease is associated with disruption in thiamin transport physiology: A potential role for neuroinflammation.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by Amyloid-ß peptide (Aß) containing plaques and cognitive deficits. The pathophysiology of AD also involves neuroinflammation. Vitamin B1 (thiamin) is indispensable for normal cellular energy metabolism. Thiamin homeostasis is altered in AD, and its deficiency is known to aggravate AD pathology. Little, however, is known about possible alterations in level of expression of thiamin transporters-1 and -2 (THTR-1 and -2) in the brain of AD, and whether pro-inflammatory cytokines affect thiamin uptake by brain cells. We addressed these issues using brain tissue samples [prefrontal cortex (PFC) and hippocampus (HIP)] from AD patients and from 5XFAD mouse model of AD, together with cultured human neuroblastoma SH-SY5Y cells as model. Our results revealed a significantly lower expression of both THTR-1 and THTR-2 in the PFC and HIP of AD patients and 5XFAD mouse model of AD compared to appropriate normal controls. Further, we found that exposure of the SH-SY5Y cells to pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α) led to a significant inhibition in thiamin uptake. Focusing on IL-1ß, we found the inhibition in thiamin uptake to be time-dependent and reversible; it was also associated with a substantial reduction in expression of THTR-1 (but not THTR-2) protein and mRNA as well as a decrease in promoter activity of the SLC19A2 gene (which encodes THTR-1). Finally, using transcriptomic analysis, we found that thiamin availability in SH-SY5Y cells caused changes in the expression of genes relevant to AD pathways. These studies demonstrate, for the first time, that thiamin transport physiology/molecular biology parameters are negatively impacted in AD brain and that pro-inflammatory cytokines inhibit thiamin uptake by neuroblastoma cells. The results also support a possible role for thiamin in the pathophysiology of AD.

Large-scale analysis reveals spatiotemporal circadian patterns of cilia transcriptomes in the primate brain.

Cilia are dynamic subcellular systems, with core structural and functional components operating in a highly coordinated manner. Since many environmental stimuli sensed by cilia are circadian in nature, it is reasonable to speculate that genes encoding cilia structural and functional components follow rhythmic circadian patterns of expression. Using computational methods and the largest spatiotemporal gene expression atlas of primates, we identified and analyzed the circadian rhythmic expression of cilia genes across 22 primate brain areas. We found that around 73% of cilia transcripts exhibited circadian rhythmicity across at least one of 22 brain regions. In 12 brain regions, cilia transcriptomes were significantly enriched with circadian oscillating transcripts, as compared to the rest of the transcriptome. The phase of the cilia circadian transcripts deviated from the phase of the majority of the background circadian transcripts, and transcripts coding for cilia basal body components accounted for the majority of cilia circadian transcripts. In addition, adjacent or functionally connected brain nuclei had large overlapping complements of circadian cilia genes. Most remarkably, cilia circadian transcripts shared across the basal ganglia nuclei and the prefrontal cortex peaked in these structures in sequential fashion that is similar to the sequential order of activation of the basal ganglia-cortical circuitry in connection with movement coordination, albeit on completely different timescales. These findings support a role for the circadian spatiotemporal orchestration of cilia gene expression in the normal physiology of the basal ganglia-cortical circuit and motor control. Studying orchestrated cilia rhythmicity in the basal ganglia-cortical circuits and other brain circuits may help develop better functional models, and shed light on the causal effects cilia functions have on these circuits and on the regulation of movement and other behaviors.

Dynamic Changes of Brain Cilia Transcriptomes across the Human Lifespan.

Almost all brain cells contain primary cilia, antennae-like microtubule sensory organelles, on their surface, which play critical roles in brain functions. During neurodevelopmental stages, cilia are essential for brain formation and maturation. In the adult brain, cilia play vital roles as signaling hubs that receive and transduce various signals and regulate cell-to-cell communications. These distinct roles suggest that cilia functions, and probably structures, change throughout the human lifespan. To further understand the age-dependent changes in cilia roles, we identified and analyzed age-dependent patterns of expression of cilia's structural and functional components across the human lifespan. We acquired cilia transcriptomic data for 16 brain regions from the BrainSpan Atlas and analyzed the age-dependent expression patterns using a linear regression model by calculating the regression coefficient. We found that 67% of cilia transcripts were differentially expressed genes with age (DEGAs) in at least one brain region. The age-dependent expression was region-specific, with the highest and lowest numbers of DEGAs expressed in the ventrolateral prefrontal cortex and hippocampus, respectively. The majority of cilia DEGAs displayed upregulation with age in most of the brain regions. The transcripts encoding cilia basal body components formed the majority of cilia DEGAs, and adjacent cerebral cortices exhibited large overlapping pairs of cilia DEGAs. Most remarkably, specific α/ß-tubulin subunits (TUBA1A, TUBB2A, and TUBB2B) and SNAP-25 exhibited the highest rates of downregulation and upregulation, respectively, across age in almost all brain regions. α/ß-tubulins and SNAP-25 expressions are known to be dysregulated in age-related neurodevelopmental and neurodegenerative disorders. Our results support a role for the high dynamics of cilia structural and functional components across the lifespan in the normal physiology of brain circuits. Furthermore, they suggest a crucial role for cilia signaling in the pathophysiological mechanisms of age-related psychiatric/neurological disorders.

Electrochemical Micropyramid Array-Based Sensor for In Situ Monitoring of Dopamine Released from Neuroblastoma Cells.

Abnormal dopamine neurotransmission is associated with several neurological and psychiatric disorders such as Parkinson's disease, schizophrenia, attention deficiency and hyperactivity disorder, and addiction. Developing highly sensitive, selective, and fast dopamine monitoring methods is of high importance especially for the early diagnosis of these diseases. Herein, we report a new ultrasensitive electrochemical sensing platform for in situ monitoring of cell-secreted dopamine using Au-coated arrays of micropyramid structures integrated directly into a Petri dish. This approach enables the monitoring of dopamine released from cells in real-time without the need for relocating cultured cells. According to the electrochemical analyses, our dopamine sensing platform exhibits excellent analytical characteristics with a detection limit of 0.50 ± 0.08 nM, a wide linear range of 0.01-500 µM, and a sensitivity of 0.18 ± 0.01 µA/µM. The sensor also has remarkable selectivity toward DA in the presence of different potentially interfering small molecules. The developed electrochemical sensor has great potential for in vitro analysis of neuronal cells as well as early diagnosis of different neurological diseases related to abnormal levels of dopamine.

Microfluidic Electrochemical Sensor for Cerebrospinal Fluid and Blood Dopamine Detection in a Mouse Model of Parkinson's Disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder involving dopaminergic neurons from the substantia nigra. The loss of dopaminergic neurons results in decreased dopamine (DA) release in the striatum and thus impaired motor functions. DA is one of the key neurotransmitters monitored for the diagnosis and during the progression and treatment of PD. Therefore, sensitive and selective DA detection methods are of high clinical relevance. In this study, a new microfluidic device utilized for electrochemical DA detection is reported. The microfluidic sensing device operates in the range of 0.1-1000 nM DA requiring only ∼2.4 µL sample volume, which corresponds to detectable 240 amol of DA. Using this sensor, we were able to monitor the changes in DA levels in cerebrospinal fluid and plasma of a mouse model of PD and following the treatment of drug l-3,4-dihydroxyphenylalanine.

Melanin Concentrating Hormone Signaling Deficits in Schizophrenia: Association With Memory and Social Impairments and Abnormal Sensorimotor Gating.

BACKGROUND: Evidence from anatomical, pharmacological, and genetic studies supports a role for the neuropeptide melanin concentrating hormone system in modulating emotional and cognitive functions. Genome-wide association studies revealed a potential association between the melanin concentrating hormone receptor (MCHR1) gene locus and schizophrenia, and the largest genome-wide association study conducted to date shows a credible genome-wide association. METHODS: We analyzed MCHR1 and pro-melanin concentrating hormone RNA-Seq expression in the prefrontal cortex in schizophrenia patients and healthy controls. Disruptions in the melanin concentrating hormone system were modeled in the mouse brain by germline deletion of MCHR1 and by conditional ablation of melanin concentrating hormone expressing neurons using a Cre-inducible diphtheria toxin system. RESULTS: MCHR1 expression is decreased in the prefrontal cortex of schizophrenia samples (false discovery rate (FDR) P < .05, CommonMind and PsychEncode combined datasets, n = 901) while pro-melanin concentrating hormone is below the detection threshold. MCHR1 expression decreased with aging (P = 6.6E-57) in human dorsolateral prefrontal cortex. The deletion of MCHR1 was found to lead to behavioral abnormalities mimicking schizophrenia-like phenotypes: hyperactivity, increased stereotypic and repetitive behavior, social impairment, impaired sensorimotor gating, and disrupted cognitive functions. Conditional ablation of pro-melanin concentrating hormone neurons increased repetitive behavior and produced a deficit in sensorimotor gating. CONCLUSIONS: Our study indicates that early disruption of the melanin concentrating hormone system interferes with neurodevelopmental processes, which may contribute to the pathogenesis of schizophrenia. Further neurobiological research on the developmental timing and circuits that are affected by melanin concentrating hormone may lead to a therapeutic target for early prevention of schizophrenia.

Receptor-specific crosstalk between prostanoid E receptor 3 and bombesin receptor subtype 3.

Bombesin receptor subtype 3 (BRS-3) is a GPCR that is expressed in the CNS, peripheral tissues, and tumors. Our understanding of BRS-3's role in physiology and pathophysiology is limited because its natural ligand is unknown. In an attempt to identify this ligand, we screened toad skin ( Bufo bufo gargarizans Cantor) extracts and identified prostaglandins as putative ligands. In BRS-3-transfected human embryonic kidney (HEK) cells, we found that prostaglandins, with prostaglandin E2 (PGE2) being the most potent, fulfill the pharmacologic criteria of affinity, selectivity, and specificity to be considered as agonists to the BRS-3 receptor. However, PGE2 is unable to activate BRS-3 in different cellular environments. We speculated that EP receptors might be the cause of this cellular selectivity, and we found that EP3 is the receptor primarily responsible for the differential PGE2 effect. Consequently, we reconstituted the HEK environment in Chinese hamster ovary (CHO) cells and found that BRS-3 and EP3 interact to potentiate PGE2 signaling. This potentiating effect is receptor specific, and it occurs only when BRS-3 is paired to EP3. Our study represents an example of functional crosstalk between two distantly related GPCRs and may be of clinical importance for BRS-3-targeted therapies.-Zhang, Y., Liu, Y., Wu, L., Fan, C., Wang, Z., Zhang, X., Alachkar, A., Liang, X., Civelli, O. Receptor-specific crosstalk between prostanoid E receptor 3 and bombesin receptor subtype 3.

A Natural Product with High Affinity to Sigma and 5-HT7 Receptors as Novel Therapeutic Drug for Negative and Cognitive Symptoms of Schizophrenia.

Dehydrocorybulbine (DHCB), an alkaloid from Corydalis yanhusuo. W.T, has been identified as a dopamine receptor antagonist. We extended our assessment of its pharmacological profile and found that DHCB exhibits high to moderate binding affinities to sigma 1 and 2 receptors, serotonin 5-HT7 receptor, and histamine H2 receptors. This led us to evaluate DHCB properties in pharmacological (apomorphine and MK-801) animal models of schizophrenia in mice. The pharmacological profile of DHCB was screened through radioligand receptor binding assays. Single dose of DHCB reversed the locomotor hyperactivity, stereotypy, and prepulse inhibition deficits induced by the dopaminergic agonist apomorphine. DHCB also reversed the depressive-like behavior and memory deficit induced by the glutamatergic antagonist MK-801 in the forced swim and the novel object recognition assays, respectively. These results indicate that DHCB effectively improves schizophrenia-like behavioral deficits that are induced by the disruption of dopaminergic and glutamatergic systems. The effectiveness of DHCB in reversing responses that mimic negative and cognitive deficits of schizophrenia might suggest that its anti-schizophrenia effects are mediated through modulating the activities of several receptor particularly sigma 1, sigma 2, 5-HT7 and dopamine receptors. Our study casts DHCB as a promising lead for therapeutic treatment of schizophrenia.

A Methionine-Induced Animal Model of Schizophrenia: Face and Predictive Validity.

BACKGROUND: Modulating the methylation process induces broad biochemical changes, some of which may be involved in schizophrenia. Methylation is in particular central to epigenesis, which is also recognized as a factor in the etiology of schizophrenia. Because methionine administration to patients with schizophrenia has been reported to exacerbate their psychotic symptoms and because mice treated with methionine exhibited social deficits and prepulse inhibition impairment, we investigated whether methionine administration could lead to behavioral changes that reflect schizophrenic symptoms in mice. METHODS: l-Methionine was administered to mice twice a day for 7 days. RESULTS: We found that this treatment induces behavioral responses that reflect the 3 types of schizophrenia-like symptoms (positive, negative, or cognitive deficits) as monitored in a battery of behavioral assays (locomotion, stereotypy, social interaction, forced swimming, prepulse inhibition, novel object recognition, and inhibitory avoidance). Moreover, these responses were differentially reversed by typical haloperidol and atypical clozapine antipsychotics in ways that parallel their effects in schizophrenics. CONCLUSION: We thus propose the l-methionine treatment as an animal model recapitulating several symptoms of schizophrenia. We have established the face and predictive validity for this model. Our model relies on an essential natural amino acid and on an intervention that is relatively simple and time effective and may offer an additional tool for assessing novel antipsychotics.

Association between polymorphisms in apoptotic genes and susceptibility for developing breast cancer in Syrian women.

Apoptosis is a major protective mechanism against cancer. The tumor suppressor protein p53 is the central protein in the apoptotic pathway and was shown to harbor mutations in a considerable fraction of breast cancer tumors. The NQO1 was shown to act as a p53 stabilizer and was suggested to play an important role in the protection against carcinogenic catechol estrogens. Functional polymorphisms in TP53 and NQO1 were investigated in relation to breast cancer susceptibility in several studies, primarily involving Asian and Caucasian populations. The aim of the present study was to investigate TP53 and NQO1 polymorphisms and their combined effects with respect to breast cancer susceptibility in a Syrian study cohort. The study cohort consisted of 122 cases and 139 controls. The tetra-primer ARMS-PCR method was used to genotype three TP53 polymorphisms; namely, exon 4 G>C Arg72Pro, IVS3 16 bp Del/Ins, and MspI IVS6+62A>G, and NQO1 C609T (Pro187Ser) polymorphism. Association was tested under six genetic models. We found a significant association for the heterozygous Arg/Pro genotype when combined with heterozygosity for IVS3 16 bp Del/Ins and MspI IVS6+62A>G (OR = 2.05 (1.22-3.47), P = 0.006). No significant association was found for NQO1 C609T or its combinations with TP53 polymorphisms. Our results support an association for TP53 polymorphisms with breast cancer susceptibility in the Syrian population.

Cilia in the Striatum Mediate Timing-Dependent Functions.

Almost all brain cells contain cilia, antennae-like microtubule-based organelles. Yet, the significance of cilia, once considered vestigial organelles, in the higher-order brain functions is unknown. Cilia act as a hub that senses and transduces environmental sensory stimuli to generate an appropriate cellular response. Similarly, the striatum, a brain structure enriched in cilia, functions as a hub that receives and integrates various types of environmental information to drive appropriate motor response. To understand cilia's role in the striatum functions, we used loxP/Cre technology to ablate cilia from the dorsal striatum of male mice and monitored the behavioral consequences. Our results revealed an essential role for striatal cilia in the acquisition and brief storage of information, including learning new motor skills, but not in long-term consolidation of information or maintaining habitual/learned motor skills. A fundamental aspect of all disrupted functions was the "time perception/judgment deficit." Furthermore, the observed behavioral deficits form a cluster pertaining to clinical manifestations overlapping across psychiatric disorders that involve the striatum functions and are known to exhibit timing deficits. Thus, striatal cilia may act as a calibrator of the timing functions of the basal ganglia-cortical circuit by maintaining proper timing perception. Our findings suggest that dysfunctional cilia may contribute to the pathophysiology of neuro-psychiatric disorders, as related to deficits in timing perception.

Behavioral and neuro-functional consequences of eliminating the KCNQ3 GABA binding site in mice.

Voltage-gated potassium (Kv) channels formed by α subunits KCNQ2-5 are important in regulating neuronal excitability. We previously found that GABA directly binds to and activates channels containing KCNQ3, challenging the traditional understanding of inhibitory neurotransmission. To investigate the functional significance and behavioral role of this direct interaction, mice with a mutated KCNQ3 GABA binding site (Kcnq3-W266L) were generated and subjected to behavioral studies. Kcnq3-W266L mice exhibited distinctive behavioral phenotypes, of which reduced nociceptive and stress responses were profound and sex-specific. In female Kcnq3-W266L mice, the phenotype was shifted towards more nociceptive effects, while in male Kcnq3-W266L mice, it was shifted towards the stress response. In addition, female Kcnq3-W266L mice exhibited lower motor activity and reduced working spatial memory. The neuronal activity in the lateral habenula and visual cortex was altered in the female Kcnq3-W266L mice, suggesting that GABAergic activation of KCNQ3 in these regions may play a role in the regulation of the responses. Given the known overlap between the nociceptive and stress brain circuits, our data provide new insights into a sex-dependent role of KCNQ3 in regulating neural circuits involved in nociception and stress, via its GABA binding site. These findings identify new targets for effective treatments for neurological and psychiatric conditions such as pain and anxiety.

Untargeted metabolomics analysis on kidney tissues from mice reveals potential hypoxia biomarkers.

Chronic hypoxia may have a huge impact on the cardiovascular and renal systems. Advancements in microscopy, metabolomics, and bioinformatics provide opportunities to identify new biomarkers. In this study, we aimed at elucidating the metabolic alterations in kidney tissues induced by chronic hypoxia using untargeted metabolomic analyses. Reverse phase ultrahigh performance liquid chromatography-mass spectroscopy/mass spectroscopy (RP-UPLC-MS/MS) and hydrophilic interaction liquid chromatography (HILIC)-UPLC-MS/MS methods with positive and negative ion mode electrospray ionization were used for metabolic profiling. The metabolomic profiling revealed an increase in metabolites related to carnitine synthesis and purine metabolism. Additionally, there was a notable increase in bilirubin. Heme, N-acetyl-L-aspartic acid, thyroxine, and 3-beta-Hydroxy-5-cholestenoate were found to be significantly downregulated. 3-beta-Hydroxy-5-cholestenoate was downregulated more significantly in male than female kidneys. Trichome Staining also showed remarkable kidney fibrosis in mice subjected to chronic hypoxia. Our study offers potential intracellular metabolite signatures for hypoxic kidneys.

Reanalysis of primate brain circadian transcriptomics reveals connectivity-related oscillations.

Research shows that brain circuits controlling vital physiological processes are closely linked with endogenous time-keeping systems. In this study, we aimed to examine oscillatory gene expression patterns of well-characterized neuronal circuits by reanalyzing publicly available transcriptomic data from a spatiotemporal gene expression atlas of a non-human primate. Unexpectedly, brain structures known for regulating circadian processes (e.g., hypothalamic nuclei) did not exhibit robust cycling expression. In contrast, basal ganglia nuclei, not typically associated with circadian physiology, displayed the most dynamic cycling behavior of its genes marked by sharp temporally defined expression peaks. Intriguingly, the mammillary bodies, considered hypothalamic nuclei, exhibited gene expression patterns resembling the basal ganglia, prompting reevaluation of their classification. Our results emphasize the potential for high throughput circadian gene expression analysis to deepen our understanding of the functional synchronization across brain structures that influence physiological processes and resulting complex behaviors.

Spatiotemporal Mapping of Brain Cilia Reveals Region-Specific Oscillation of Length and Orientation.

In this study, we conducted high-throughput spatiotemporal analysis of primary cilia length and orientation across 22 mouse brain regions. We developed automated image analysis algorithms, which enabled us to examine over 10 million individual cilia, generating the largest spatiotemporal atlas of cilia. We found that cilia length and orientation display substantial variations across different brain regions and exhibit fluctuations over a 24-hour period, with region-specific peaks during light-dark phases. Our analysis revealed unique orientation patterns of cilia at 45 degree intervals, suggesting that cilia orientation within the brain is not random but follows specific patterns. Using BioCycle, we identified circadian rhythms of cilia length in five brain regions: nucleus accumbens core, somatosensory cortex, and three hypothalamic nuclei. Our findings present novel insights into the complex relationship between cilia dynamics, circadian rhythms, and brain function, highlighting cilia crucial role in the brain's response to environmental changes and regulation of time-dependent physiological processes.

Association of polymorphisms in one-carbon metabolizing genes with breast cancer risk in Syrian women.

Dietary folate status as well as polymorphisms in one-carbon metabolism genes may affect the risk of breast cancer through aberrant DNA methylation and altered nucleotide synthesis and DNA repair. A large number of studies investigated the role of methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) polymorphisms in breast cancer with inconsistent results. Association between multiple polymorphisms in one-carbon metabolism genes and breast cancer was not studied before in an Arab population. The purpose of the present study is to test the hypothesis that polymorphisms in one-carbon metabolism genes are associated with breast cancer susceptibility in Syrian breast cancer women patients. A total of 245 subjects (119 breast cancer women patients and 126 healthy controls) were genotyped for MTHFR C677T and A1298C and MTRR A66G polymorphisms. Association was tested for under numerous genetic models. A statistically significant association was found for MTHFR A1298C polymorphism especially under the allele contrast model (odds ratio (OR) = 1.68, 95% confidence interval (CI) (1.16-2.45), P = 0.006). On the other hand, no significant association was found for MTHFR C677T or MTRR A66G under any of the genetic models tested. The effects of the compound genotypes were also examined. The 66GG genotype was found to be protective against breast cancer when combined with the 677CT or 1298AC genotype (OR = 0.18, 95% CI (0.04-0.82), P = 0.014; OR = 0.3, 95% CI (0.08-1.11), P = 0.058). In conclusion, our study supports the hypothesis that polymorphisms in one-carbon gene metabolisms modulate the risk for breast cancer, particularly the A1298C polymorphism of the MTHFR gene.

Triplex tetra-primer ARMS-PCR method for the simultaneous detection of MTHFR c.677C>T and c.1298A>C, and MTRR c.66A>G polymorphisms of the folate-homocysteine metabolic pathway.

The folate-homocysteine metabolic pathway was shown to play an important role in several diseases such as cancers, cardiovascular diseases, and neurodegenerative diseases. The c.677C>T and c.1298A>C polymorphisms of the Methylenetetrahydrofolate reductase (MTHFR) gene, and c.66A>G of the Methionine synthase reductase (MTRR) gene are the most commonly investigated polymorphisms in the folate-homocysteine metabolic pathway. The currently used methods for the detection of the three polymorphisms are either slow and laborious or extremely expensive. In this paper, a new highly optimized method for the simultaneous detection of the three single nucleotide polymorphisms is described. The proposed method utilizes 12 primers in a single PCR reaction to detect the three polymorphisms simultaneously based on the principle of tetra-primer ARMS-PCR (also known as PCR-CTPP). The proposed method offers extremely fast, economical, and simple detection. Validation by PCR-RFLP showed 100% concordance in genotype assignment. The proposed method was successfully applied to a sample of the Syrian population (n=126), which was not previously genotyped for any of the three SNPs. The variant allele frequencies were found to be 31, 29, and 43% for the c.677C>T, c.1298A>C, and c.66A>G polymorphisms, respectively. The proposed method is the first to detect three SNPs in a single PCR reaction based on tetra-primer ARMS-PCR or PCR-CTPP. We suggest that the use of Betaine may play an important role in multiplex tetra-primer ARMS-PCR or PCR-CTPP based on its potential capacity to close the gap in melting temperature between different primers.

Aromatic patterns: Tryptophan aromaticity as a catalyst for the emergence of life and rise of consciousness.

Sunlight held the key to the origin of life on Earth. The earliest life forms, cyanobacteria, captured the sunlight to generate energy through photosynthesis. Life on Earth evolved in accordance with the circadian rhythms tied to sensitivity to sunlight patterns. A unique feature of cyanobacterial photosynthetic proteins and circadian rhythms' molecules, and later of nearly all photon-sensing molecules throughout evolution, is that the aromatic amino acid tryptophan (Trp) resides at the center of light-harvesting active sites. In this perspective, I review the literature and integrate evidence from different scientific fields to explore the role Trp plays in photon-sensing capabilities of living organisms through its resonance delocalization of π-electrons. The observations presented here are the product of apparently unrelated phenomena throughout evolution, but nevertheless share consistent patterns of photon-sensing by Trp-containing and Trp-derived molecules. I posit the unique capacity to transfer electrons during photosynthesis in the earliest life forms is conferred to Trp due to its aromaticity. I propose this ability evolved to assume more complex functions, serving as a host for mechanisms underlying mental aptitudes - a concept which provides a theoretical basis for defining the neural correlates of consciousness. The argument made here is that Trp aromaticity may have allowed for the inception of the mechanistic building blocks used to fabricate complexity in higher forms of life. More specifically, Trp aromatic non-locality may have acted as a catalyst for the emergence of consciousness by instigating long-range synchronization and stabilizing the large-scale coherence of neural networks, which mediate functional brain activity. The concepts proposed in this perspective provide a conceptual foundation that invites further interdisciplinary dialogue aimed at examining and defining the role of aromaticity (beyond Trp) in the emergence of life and consciousness.