Through the looking glass: milestones on the road towards mirroring life.

Naturally occurring DNA, RNA, and proteins predominantly exist in only one enantiomeric form (homochirality). Advances in biotechnology and chemical synthesis allow the production of the respective alternate enantiomeric form, enabling access to mirror-image versions of these natural biopolymers. Exploiting the unique properties of such mirror molecules has already led to many applications, such as biostable and nonimmunogenic therapeutics or sensors. However, a 'roadblock' for unlocking the mirror world is the lack of biological systems capable of synthesizing critical building blocks including mirror oligonucleotides and oligopeptides to reducing cost and improve purity. Here, we provide an overview of the current progress, applications, and challenges of the molecular mirror world by identifying milestones towards mirroring life.

Unidirectional Lasing from Mirror-Coupled Dielectric Lattices.

This paper reports how a hybrid system composed of transparent dielectric lattices over a metal mirror can produce high-quality lattice resonances for unidirectional lasing. The enhanced electromagnetic fields are concentrated in the cladding of the periodic dielectric structures and away from the metal. Based on a mirror-image model, we reveal that such high-quality lattice resonances are governed by bound states in the continuum resulting from destructive interference. Using hexagonal arrays of titanium dioxide nanoparticles on a silica-coated silver mirror, we observed lattice resonances with quality factors of up to 2750 in the visible regime. With the lattice resonances as optical feedback and dye solution as the gain medium, we demonstrated unidirectional lasing under optical pumping, where the array size was down to 100 µm × 100 µm. Our scheme can be extended to other spectral regimes to simultaneously achieve strongly enhanced surface fields and high quality factors.

An interesting electrocardiogram caused by lead reversal.

BACKGROUND: During normal sinus rhythm, atrial depolarization is conducted from right atrium to left atrium through Bachmann's bundle, and a normal P wave axis which is measured on the frontal plane is between 0º and + 75º. The change of P wave polarity is helpful for the analysis of origin point. CASE PRESENTATION: We report a patient with negative P wave in lead I. The characteristics of QRS complex in leads V1 to V6 are helpful to preliminarily differential diagnosis. The 12-lead electrocardiogram (ECG) with correct limb leads (right arm-left arm) placement shows sinus rhythm with complete right bundle branch block (RBBB). CONCLUSIONS: The change of P wave polarity as well as characteristics of QRS complex can help identify limb-lead reversals.

Three-dimensional high-definition live tissue virtual dissection of mirror-image dextrocardia with thoracic-abdominal discordance in a fetus.

Three-dimensional virtual dissection using high-definition live tissue rendering ultrasound tool of a 23-week gestation fetus with situs solitus, mirror image dextrocardia, ventricular septal defect, aortic override, and pulmonary atresia.

Utility of the Angle between the Right Aortic Arch and First Branch for Detecting Double Aortic Arch via Fetal Echocardiography.

INTRODUCTION: The presence of a double aortic arch (DAA) is manifested by compressive symptoms, requiring surgery. DAA cases are classified as either complete or incomplete type. DAA and a right aortic arch with mirror image branching (mRAA) have a similar configuration to the first branch artery. The first branch of the mRAA is the left brachiocephalic artery, which appears to be the same as that of an incomplete DAA due to blood flow interruption. The present retrospective study aimed to evaluate the differences between DAA and mRAA by fetal echocardiography. METHODS: This single retrospective cohort study included all patients diagnosed with complete DAA, incomplete DAA, or mRAA at our facility between 2010 and 2022. The patients were diagnosed with complete DAA, incomplete DAA, or mRAA after birth and remaining fetal echocardiograms. The patients were divided into the DAA (complete DAA: n = 4, incomplete DAA: n = 3) and mRAA (n = 4) groups. The following three outcomes were compared: (1) angle between the right aortic arch and first branch (RF angle), (2) ratio of height to width of the region bounded by the aortic arch, first branch of the aortic arch, and descending aorta, and (3) maximum tracheal diameter on a three-vessel trachea view. RESULTS: The incomplete DAA cases were difficult to diagnose via fetal echocardiography. On fetal echocardiography, the RF angle was significantly steeper in the DAA group than in the mRAA group (median 57° [36°-69°] vs. 75° [62°-94°]; p < 0.05). The DAA and RAA groups showed no significant differences in the ratio of height to width of the region bounded by the aortic arch, first branch of the aortic arch, and descending aorta (median 0.57 [0.17-0.68] vs. 0.73 [0.56-1.0]) and maximum tracheal diameter (median 2.5 [1.4-3.3] vs. 3.2 [2.8-3.5] mm). The cut-off value for the presence of DAA was an RF angle <71°. CONCLUSION: The DAA group (complete and incomplete DAA) had a significantly steeper RF angle than the mRAA group. Therefore, RF angle measurement could improve the fetal diagnosis and postnatal prognosis of DAA.

L-Glycosidase-Cleavable Natural Glycans Facilitate the Chemical Synthesis of Correctly Folded Disulfide-Bonded D-Proteins.

D-peptide ligands can be screened for therapeutic potency and enzymatic stability using synthetic mirror-image proteins (D-proteins), but efficient acquisition of these D-proteins can be hampered by the need to accomplish their in vitro folding, which often requires the formation of correctly linked disulfide bonds. Here, we report the finding that temporary installation of natural O-linked-ß-N-acetyl-D-glucosamine (O-GlcNAc) groups onto selected D-serine or D-threonine residues of the synthetic disulfide-bonded D-proteins can facilitate their folding in vitro, and that the natural glycosyl groups can be completely removed from the folded D-proteins to afford the desired chirally inverted D-protein targets using naturally occurring O-GlcNAcase. This approach enabled the efficient chemical syntheses of several important but difficult-to-fold D-proteins incorporating disulfide bonds including the mirror-image tumor necrosis factor alpha (D-TNFα) homotrimer and the mirror-image receptor-binding domain of the Omicron spike protein (D-RBD). Our work establishes the use of O-GlcNAc to facilitate D-protein synthesis and folding and proves that D-proteins bearing O-GlcNAc can be good substrates for naturally occurring O-GlcNAcase.

An Enzyme-Cleavable Solubilizing-Tag Facilitates the Chemical Synthesis of Mirror-Image Proteins.

Mirror-image proteins (D-proteins) are useful in biomedical research for purposes such as mirror-image screening for D-peptide drug discovery, but the chemical synthesis of many D-proteins is often low yielding due to the poor solubility or aggregation of their constituent peptide segments. Here, we report a Lys-C protease-cleavable solubilizing tag and its use to synthesize difficult-to-obtain D-proteins. Our tag is easily installed onto multiple amino acids such as DLys, DSer, DThr, and/or the N-terminal amino acid of hydrophobic D-peptides, is impervious to various reaction conditions, such as peptide synthesis, ligation, desulfurization, and transition metal-mediated deprotection, and yet can be completely removed by Lys-C protease under denaturing conditions to give the desired D-protein. The efficacy and practicality of the new method were exemplified in the synthesis of two challenging D-proteins: D-enantiomers of programmed cell death protein 1 IgV domain and SARS-CoV-2 envelope protein, in high yield. This work demonstrates that the enzymatic cleavage of solubilizing tags under denaturing conditions is feasible, thus paving the way for the production of more D-proteins.

Mirror-Image Random Nonstandard Peptide Integrated Discovery (MI-RaPID) Technology Yields Highly Stable and Selective Macrocyclic Peptide Inhibitors for Matrix Metallopeptidase 7.

Matrix metallopeptidase 7 (MMP7) plays a crucial role in cancer metastasis and progression, making it an attractive target for therapeutic development. However, the development of selective MMP7 inhibitors is challenging due to the conservation of active sites across various matrix metalloproteinases (MMPs). Here, we have developed mirror-image random nonstandard peptides integrated discovery (MI-RaPID) technology to discover innate protease-resistant macrocyclic peptides that specifically bind to and inhibit human MMP7. One identified macrocyclic peptide against D-MMP7, termed D20, was synthesized in its mirror-image form, D'20, consisting of 12 D-amino acids, one cyclic b-amino acid, and a thioether bond. Notably, it potently inhibited MMP7 with an IC50 value of 90 nM, and showed excellent selectivity over other MMPs with similar substrate specificity. Moreover, D'20 inhibited the migration of pancreatic cell line CFPAC-1, but had no effect on the cell proliferation and viability. D'20 exhibited excellent stability in human serum, as well as in simulated gastric and intestinal fluids. This study highlights that MI-RaPID technology can serve as a powerful tool to develop in vivo stable macrocyclic peptides for therapeutic applications.

Derivatization of Mirror-Image l-Nucleic Acids with 2'-OMe Modification for Thermal and Structural Stabilization.

To further expand the functionality and enhance the stability of mirror-image nucleic acids as advanced agents for basic research and therapeutic design, we have synthesized 2'-deoxy-2'-methoxy-l-uridine phosphoramidite and incorporated it into l-DNA and l-RNA by solid-phase synthesis quantitatively. We found that the thermostability of l-nucleic acids is dramatically improved after introducing the modifications. Moreover, we successfully crystallized both l-DNA and l-RNA duplexes containing the 2'-OMe modifications and sharing identical sequences. Crystal structure determination and analysis revealed the overall structures of the mirror-image nucleic acids, and for the first time it was possible to interpret the structural deviations caused by 2'-OMe and 2'-OH groups in the oligonucleotides, which are very similar. This novel chemical nucleic acid modification has the potential to be used to design nucleic acid-based therapeutics and materials in the future.

D-Peptide and D-Protein Technology: Recent Advances, Challenges, and Opportunities.

Total chemical protein synthesis provides access to entire D-protein enantiomers enabling unique applications in molecular biology, structural biology, and bioactive compound discovery. Key enzymes involved in the central dogma of molecular biology have been prepared in their D-enantiomeric forms facilitating the development of mirror-image life. Crystallization of a racemic mixture of L- and D-protein enantiomers provides access to high-resolution X-ray structures of polypeptides. Additionally, D-enantiomers of protein drug targets can be used in mirror-image phage display allowing discovery of non-proteolytic D-peptide ligands as lead candidates. This review discusses the unique applications of D-proteins including the synthetic challenges and opportunities.

The Development of Spatial Cognition and Its Malleability Assessed in Mass Population via a Mobile Game.

Spatial cognition is a fundamental aspect of human intelligence, but our understanding of its developmental trajectory across the life span is limited. Here, we applied game-based assessment on mobile devices to engage a large sample from China (N = 216,713) with a wide age range (from under 10 years old to above 60) in multiple participations of a mental rotation task, a typical measure of spatial cognition. We found that spatial ability developed asynchronously with its malleability. Whereas mental rotation performance peaked at the age of 28, with males performing better than females, the effect of training from repeated participation peaked at 18, probably laying the foundation for the development of spatial ability. In contrast, children showed particularly low malleability, and a follow-up experiment revealed that the underdeveloped ability of mirror-image discrimination likely hindered the malleability of spatial cognition during this period. The intermingled relation of ability and malleability illustrates dynamics in the development of spatial cognition, inviting broad research on the development of other cognitive functions.

Effect of long-acting injectable antipsychotics on 1-year hospitalization in bipolar disorder: a mirror-image study.

Long-acting injectable (LAI) antipsychotics are often used for the long-term management also of bipolar disorder (BD). Nonetheless, evidence on their effect on pragmatic outcomes such as hospitalization risk in BD is inconsistent. We carried out a mirror-image study comparing rates and number of days of hospitalization, one year before and after the initiation of LAI treatment, in a sample of subjects with BD. Participants were selected from the STAR Network Depot Study, a pragmatic, observational, multicenter research involving a cohort of inpatients and outpatients consecutively started on LAI treatment. Variations in rates and in total number of days of hospitalization between the 12 months before and those after treatment initiation were analyzed. Among 461 individuals screened for eligibility, we included 71 adults with BD, initiated either on first- (FGA) or second-generation (SGA) LAIs. We found a significant decrease in terms of 12-month hospitalization rates (p < 0.001) and number of days (p < 0.001) after LAI initiation, without any effect by age, gender, alcohol/substance use disorders, and symptom severity. Subgroup analyses based on antipsychotic class, history of LAI treatment, and concomitant oral medications, confirmed the decreasing trend on both hospitalization rates and number of days. However, these reductions were not significant among participants who continued this treatment for less than 6 months. Comprehensively, this study supports the role of LAIs as effective maintenance treatment options for BD. Further research is needed to identify clinical characteristics of people with BD who would most benefit from long-acting formulations of antipsychotics.

Evidence for aggregation-independent, PrPC-mediated Aß cellular internalization.

Evidence linking amyloid beta (Aß) cellular uptake and toxicity has burgeoned, and mechanisms underlying this association are subjects of active research. Two major, interconnected questions are whether Aß uptake is aggregation-dependent and whether it is sequence-specific. We recently reported that the neuronal uptake of Aß depends significantly on peptide chirality, suggesting that the process is predominantly receptor-mediated. Over the past decade, the cellular prion protein (PrPC) has emerged as an important mediator of Aß-induced toxicity and of neuronal Aß internalization. Here, we report that the soluble, nonfibrillizing Aß (1-30) peptide recapitulates full-length Aß stereoselective cellular uptake, allowing us to decouple aggregation from cellular, receptor-mediated internalization. Moreover, we found that Aß (1-30) uptake is also dependent on PrPC expression. NMR-based molecular-level characterization identified the docking site on PrPC that underlies the stereoselective binding of Aß (1-30). Our findings therefore identify a specific sequence within Aß that is responsible for the recognition of the peptide by PrPC, as well as PrPC-dependent cellular uptake. Further uptake stereodifferentiation in PrPC-free cells points toward additional receptor-mediated interactions as likely contributors for Aß cellular internalization. Taken together, our results highlight the potential of targeting cellular surface receptors to inhibit Aß cellular uptake as an alternative route for future therapeutic development for Alzheimer's disease.

Measuring "Where": A Comparative Analysis of Methods Measuring Spatial Perception.

The literature offers various methods for measuring sound localization. In this study, we aimed to compare these methods to determine their effectiveness in addressing different research questions by examining the effect sizes obtained from each measure. Data from 150 participants who identified the location of a sound source were analyzed to explore the effects of speaker angle, stimuli, HPD type, and condition (with/without HPD) on sound localization, using six methods for analysis: mean absolute deviation (MAD), root-mean-squared error (RMSE), very large errors (VLE), percentage of errors larger than the average error observed in a group of participants (pMean), percentage of errors larger than half the distance between two consecutive loudspeakers (pHalf), and mirror image reversal errors (MIRE). Results indicated that the MIRE measure was the most sensitive to the effects of speaker angle and HPD type, while the VLE measure was most sensitive to the effect of stimuli type. The condition variable provided the largest effect sizes, with no difference observed between measures. The data suggest that when effect sizes are substantial, all methods are adequate. However, for cases where the effect size is expected to be small, methods that yield larger effect sizes should be considered, considering their alignment with the research question.

Stabilization of Monomeric Tau Protein by All D-Enantiomeric Peptide Ligands as Therapeutic Strategy for Alzheimer's Disease and Other Tauopathies.

Alzheimer's disease and other tauopathies are the world's leading causes of dementia and memory loss. These diseases are thought to be caused by the misfolding and aggregation of the intracellular tau protein, ultimately leading to neurodegeneration. The tau protein is involved in a multitude of different neurodegenerative diseases. During the onset of tauopathies, tau undergoes structural changes and posttranslational modifications and aggregates into amyloid fibrils that are able to spread with a prion-like behavior. Up to now, there is no therapeutic agent which effectively controls or reverses the disease. Most of the therapeutics that were developed and underwent clinical trials targeted misfolded or aggregated forms of tau. In the current manuscript, we present the selection and characterization of two all D-enantiomeric peptides that bind monomeric tau protein with a low nanomolar KD, stabilize tau in its monomeric intrinsically disordered conformation, and stop the conversion of monomers into aggregates. We show that the effect of the two all D-enantiomeric peptides is strong enough to stop ongoing tau aggregation in vitro and is able to significantly reduce tau fibril assembly in cell culture. Both compounds may serve as new lead components for the development of therapeutic agents against Alzheimer's disease and other tauopathies.

Backbone-Installed Split Intein-Assisted Ligation for the Chemical Synthesis of Mirror-Image Proteins.

Membrane-associated D-proteins are an important class of synthetic molecules needed for D-peptide drug discovery, but their chemical synthesis using canonical ligation methods such as native chemical ligation is often hampered by the poor solubility of their constituent peptide segments. Here, we describe a Backbone-Installed Split Intein-Assisted Ligation (BISIAL) method for the synthesis of these proteins, wherein the native L-forms of the N- and C-intein fragments of the unique consensus-fast (Cfa) (i.e. L-CfaN and L-CfaC ) are separately installed onto the two D-peptide segments to be ligated via a removable backbone modification. The ligation proceeds smoothly at micromolar (µM) concentrations under strongly chaotropic conditions (8.0 M urea), and the subsequent removal of the backbone modification groups affords the desired D-proteins without leaving any "ligation scar" on the products. The effectiveness and practicality of the BISIAL method are exemplified by the synthesis of the D-enantiomers of the extracellular domains of T cell immunoglobulin and ITIM domain (TIGIT) and tropomyosin receptor kinase C (TrkC). The BISIAL method further expands the chemical protein synthesis ligation toolkit and provides practical access to challenging D-protein targets.

Contralateral Projection of Anterior Cingulate Cortex Contributes to Mirror-Image Pain.

Long-term limb nerve injury often leads to mirror-image pain (MIP), an abnormal pain sensation in the limb contralateral to the injury. Although it is clear that MIP is mediated in part by central nociception processing, the underlying mechanisms remain poorly understood. The anterior cingulate cortex (ACC) is a key brain region that receives relayed peripheral nociceptive information from the contralateral limb. In this study, we induced MIP in male mice, in which a unilateral chronic constrictive injury of the sciatic nerve (CCI) induced a decreased nociceptive threshold in both hind limbs and an increased number of c-Fos-expressing neurons in the ACC both contralateral and ipsilateral to the injured limb. Using viral-mediated projection mapping, we observed that a portion of ACC neurons formed monosynaptic connections with contralateral ACC neurons. Furthermore, the number of cross-callosal projection ACC neurons that exhibited c-Fos signal was increased in MIP-expressing mice, suggesting enhanced transmission between ACC neurons of the two hemispheres. Moreover, selective inhibition of the cross-callosal projection ACC neurons contralateral to the injured limb normalized the nociceptive sensation of the uninjured limb without affecting the increased nociceptive sensation of the injured limb in CCI mice. In contrast, inhibition of the non-cross-callosal projection ACC neurons contralateral to the injury normalized the nociceptive sensation of the injured limb without affecting the MIP exhibited in the uninjured limb. These results reveal a circuit mechanism, namely, the cross-callosal projection of ACC between two hemispheres, that contributes to MIP and possibly other forms of contralateral migration of pain sensation.SIGNIFICANCE STATEMENT Mirror-image pain (MIP) refers to the increased pain sensitivity of the contralateral body part in patients with chronic pain. This pathology requires central processing, yet the mechanisms are less known. Here, we demonstrate that the cross-callosal projection neurons in the anterior cingulate cortex (ACC) contralateral to the injury contribute to MIP exhibited in the uninjured limb, but do not affect nociceptive sensation of the injured limb. In contrast, the non-cross-callosal projection neurons in the ACC contralateral to the injury contribute to nociceptive sensation of the injured limb, but do not affect MIP exhibited in the uninjured limb. Our study depicts a novel cross-callosal projection of ACC that contributes to MIP, providing a central mechanism for MIP in chronic pain state.

Complete Freund's adjuvant-induced protein dysregulation correlated with mirror image pain as assessed by quantitative proteomics of the mouse spinal cord.

Inflammation or trauma occurring on one side of the body can cause pathological pain on the contralateral noninjured side in a phenomenon called mirror-image pain (MIP). Although some potential mechanisms involved in MIP have been reported, including those involving the immune system and glial cells as well as neural mechanisms, the molecular mechanisms are not well understood. In this study, we aimed to understand the molecular mechanisms in MIP using quantitative proteomics and whole-cell patch clamp recordings. Behavioral test results showed that complete Freund's adjuvant could induce MIP in the mice. The results of isobaric tags for relative and absolute quantification (iTRAQ) quantitative proteomics showed that 108 proteins were dysregulated, and these proteins may represent potential targets. Furthermore, bioinformatics analysis was applied to explore the potential molecular mechanisms during MIP after complete Freund's adjuvant (CFA) treatment. Parallel reaction monitoring (PRM) results showed that PKCδ and seven other dysregulated proteins were related to MIP after CFA treatment. Patch clamp recording results showed that CFA treatment could increase intrinsic excitability and spontaneous firing in spinal cord neurons during MIP. In summary, we found that CFA could induce MIP. The results of proteomic research on the spinal cord after CFA treatment could provide new insight into the molecular mechanisms of MIP. Moreover, the neuronal activity of spinal cord neurons was upregulated during MIP after CFA treatment. In summary, the results of the spinal cord proteomic profile provide a potential molecular mechanism for understanding MIP.

Neuroinflammation in the anterior cingulate cortex: the potential supraspinal mechanism underlying the mirror-image pain following motor fiber injury.

BACKGROUND: Peripheral nerve inflammation or lesion can affect contralateral healthy structures, and thus result in mirror-image pain. Supraspinal structures play important roles in the occurrence of mirror pain. The anterior cingulate cortex (ACC) is a first-order cortical region that responds to painful stimuli. In the present study, we systematically investigate and compare the neuroimmune changes in the bilateral ACC region using unilateral- (spared nerve injury, SNI) and mirror-(L5 ventral root transection, L5-VRT) pain models, aiming to explore the potential supraspinal neuroimmune mechanism underlying the mirror-image pain. METHODS: The up-and-down method with von Frey hairs was used to measure the mechanical allodynia. Viral injections for the designer receptors exclusively activated by designer drugs (DREADD) were used to modulate ACC glutamatergic neurons. Immunohistochemistry, immunofluorescence, western blotting, protein microarray were used to detect the regulation of inflammatory signaling. RESULTS: Increased expressions of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and chemokine CX3CL1 in ACC induced by unilateral nerve injury were observed on the contralateral side in the SNI group but on the bilateral side in the L5-VRT group, representing a stronger immune response to L5-VRT surgery. In remote ACC, both SNI and L5-VRT induced robust bilateral increase in the protein level of Nav1.6 (SCN8A), a major voltage-gated sodium channel (VGSC) that regulates neuronal activity in the mammalian nervous system. However, the L5-VRT-induced Nav1.6 response occurred at PO 3d, earlier than the SNI-induced one, 7 days after surgery. Modulating ACC glutamatergic neurons via DREADD-Gq or DREADD-Gi greatly changed the ACC CX3CL1 levels and the mechanical paw withdrawal threshold. Neutralization of endogenous ACC CX3CL1 by contralateral anti-CX3CL1 antibody attenuated the induction and the maintenance of mechanical allodynia and eliminated the upregulation of CX3CL1, TNF-α and Nav1.6 protein levels in ACC induced by SNI. Furthermore, contralateral ACC anti-CX3CL1 also inhibited the expression of ipsilateral spinal c-Fos, Iba1, CD11b, TNF-α and IL-6. CONCLUSIONS: The descending facilitation function mediated by CX3CL1 and its downstream cascade may play a pivotal role, leading to enhanced pain sensitization and even mirror-image pain. Strategies that target chemokine-mediated ACC hyperexcitability may lead to novel therapies for the treatment of neuropathic pain.

Reflected image processing abnormalities in dementia - a case series and review.

Dementia is a neurodegenerative condition with progressive decline in cognitive faculties and associated with different clinical phenomena. Mirror phenomenon in terms of both mirror agnosia and mirror image agnosia wherein there is difficulty in processing and perception of reflected images is not uncommonly seen, and understanding the same can contribute to early diagnosis and prognostication. We report two elderly women with Alzheimer's dementia and frontotemporal dementia, respectively, presenting with features of abnormalities in mirror image processing. The former had features of both mirror agnosia and mirror image agnosia and the latter had predominantly features of mirror image agnosia with preoccupation with her mirror image. Understanding neuroanatomical networks underlying these phenomena can help early identification and subtyping dementia. Clinically differentiating these phenomena from psychotic symptoms of dementia can help in initiating appropriate non-pharmacological measures rather than resorting to use of psychotropics, the use of which may be counterproductive.