Scope and Limitations of Using Microemulsions for the Covalent Patterning of Graphene.

Patterning of graphene (functionalizing some areas while leaving others intact) is challenging, as all the C atoms in the basal plane are identical, but it is also desirable for a variety of applications, like opening a bandgap in the electronic structure of graphene. Several methods have been reported to pattern graphene, but most of them are very technologically intensive. Recently, we reported the use of microemulsions as templates to pattern graphene at the µm scale. This method is very simple and in principle tunable, as emulsions of different droplet size and composition can be prepared easily. Here, we explore in detail the scope of this methodology by applying it to all the combinations of four different emulsions and three different organic reagents, and characterizing the resulting substrates exhaustively through Raman, SEM and AFM. We find that the method is general, works better when the reactive species are outside the micelles, and requires reactive species that involve short reaction times.

Control over Dethreading Kinetics Allows Evaluating the Entropy Stored in an Interlocked Molecular Machine Out-of-Equilibrium.

The operation of nanomachines is fundamentally different from that of their macroscopic counterparts. In particular, the role of solvent is critical yet rarely associated with machine functionality. Here, we study a minimal model of one of the most advanced molecular machines to gain control of its operation by engineering components and the employed solvent. Operation kinetics were changed over more than four orders of magnitude and could be modulated by solvent. Leveraging solvent properties, it was possible to monitor the relaxation of the molecular machine towards equilibrium and measure the heat exchanged in the process. Our work expands the capabilities of acid-base powered molecular machines, confirming experimentally that such systems have a dominant entropy content.

The Covalent Functionalization of Surface-Supported Graphene: An Update.

In the last decade, the use of graphene supported on solid surfaces has broadened its scope and applications, and graphene has acquire a promising role as a major component of high-performance electronic devices. In this context, the chemical modification of graphene has become essential. In particular, covalent modification offers key benefits, including controllability, stability, and the facility to be integrated into manufacturing operations. In this Review, we critically comment on the latest advances in the covalent modification of supported graphene on substrates. We analyze the different chemical modifications with special attention to radical reactions. In this context, we review the latest achievements in reactivity control, tailoring electronic properties, and introducing active functionalities. Finally, we extended our analysis to other emerging 2D materials supported on surfaces, such as transition metal dichalcogenides, transition metal oxides, and elemental analogs of graphene.

Enhancing Oxygenic Photosynthesis by Cross-Linked Perylenebisimide "Quantasomes".

As the natural-born photoelectrolyzer for oxygen delivery, photosystem II (PSII) is hardly replicated with man-made constructs. However, building on the "quantasome" hypothesis ( Science 1964, 144, 1009-1011), PSII mimicry can be pared down to essentials by shaping a photocatalytic ensemble (from the Greek term "soma" = body) where visible-light quanta trigger water oxidation. PSII-inspired quantasomes (QS) readily self-assemble into hierarchical photosynthetic nanostacks, made of bis-cationic perylenebisimides (PBI2+) as chromophores and deca-anionic tetraruthenate polyoxometalates (Ru4POM) as water oxidation catalysts ( Nat. Chem. 2019, 11, 146-153). A combined supramolecular and click-chemistry strategy is used herein to interlock the PBI-QS with tetraethylene glycol (TEG) cross-linkers, yielding QS-TEGlock with increased water solvation, controlled growth, and up to a 340% enhancement of the oxygenic photocurrent compared to the first generation QS, as probed on 3D-inverse opal indium tin oxide electrodes at 8.5 sun irradiance (λ > 450 nm, 1.28 V vs RHE applied bias, TOFmax = 0.096 ± 0.005 s-1, FEO2 > 95%). Action spectra, catalyst mass-activity, light-management, photoelectrochemical impedance spectroscopy (PEIS) together with Raman mapping of TEG-templated hydration shells point to a key role of the cross-linked PBI/Ru4POM nanoarrays, where the interplay of hydrophilic/hydrophobic domains is reminiscent of PSII-rich natural thylakoids.

Mono- and Tripodal Porphyrins: Investigation on the Influence of the Number of Pyrene Anchors in Carbon Nanotube and Graphene Hybrids.

A series of molecular precursors, containing one (1 and 3) or three (2 and 4) pyrene anchors, covalently linked to porphyrins (free base or Zn), were prepared and characterized. All of them enable their π-π stacking onto low-dimensional nanocarbons including single-walled carbon nanotubes (SWCNTs) and nanographene (NG), their individualization, and their characterization. Microscopic (TEM, AFM) and spectroscopic (steady-state UV-vis and fluorescence, spectroelectrochemistry, and transient absorption measurements) techniques were at the forefront of the characterizations and were complemented by Raman spectroscopy and theoretical calculations. Of great importance is the Raman analysis, which corroborated n-doping of the nanocarbons due to the interactions with 1-4 when probed in the solid state. In solution, the situation is, however, quite different. Efficient charge separation was only observed for the graphene-based system NG/3.

The effect of an engineered ATCUN motif on the structure and biophysical properties of the SH3 domain of c-Src tyrosine kinase.

Metal binding to sites engineered in proteins can provide an increase in their stability and facilitate new functions. Besides the sites introduced in purpose, sometimes they are present accidentally as a consequence of the expression system used to produce the protein. This happens with the copper- and nickel-binding (ATCUN) motif generated by the amino-terminal residues Gly-Ser-His. This ATCUN motif is fortuitously present in many proteins, but how it affects the structural and biophysical characterization of the proteins has not been studied. In this work, we have compared the structure and biophysical properties of a small modular domain, the SH3 domain of the c-Src tyrosine kinase, cloned with and without an ATCUN motif at the N terminus. At pH 7.0, the SH3 domain with the ATCUN motif binds nickel with a binding constant Ka = 28.0 ± 3.0 mM-1. The formation of the nickel complex increases the thermal and chemical stability of the SH3 domain. A comparison of the crystal structures of the SH3 domain with and without the ATCUN motif shows that the binding of nickel does not affect the overall structure of the SH3 domain. In all crystal structures analyzed, residues Gly-Ser-His in complex with Ni2+ show a square planar geometry. The CD visible spectrum of the nickel complex shows that this geometry is also present in the solution. Therefore, our results not only show that the ATCUN motif might influence the biophysical properties of the protein, but also points to an advantageous stabilization of the protein with potential biotechnological applications.

Loss of HCN1 enhances disease progression in mouse models of CNG channel-linked retinitis pigmentosa and achromatopsia.

Most inherited blinding diseases are characterized by compromised retinal function and progressive degeneration of photoreceptors. However, the factors that affect the life span of photoreceptors in such degenerative retinal diseases are rather poorly understood. Here, we explore the role of hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1) in this context. HCN1 is known to adjust retinal function under mesopic conditions, and although it is expressed at high levels in rod and cone photoreceptor inner segments, no association with any retinal disorder has yet been found. We investigated the effects of an additional genetic deletion of HCN1 on the function and survival of photoreceptors in a mouse model of CNGB1-linked retinitis pigmentosa (RP). We found that the absence of HCN1 in Cngb1 knockout (KO) mice exacerbated photoreceptor degeneration. The deleterious effect was reduced by expression of HCN1 using a viral vector. Moreover, pharmacological inhibition of HCN1 also enhanced rod degeneration in Cngb1 KO mice. Patch-clamp recordings revealed that the membrane potentials of Cngb1 KO and Cngb1/Hcn1 double-KO rods were both significantly depolarized. We also found evidence for altered calcium homeostasis and increased activation of the protease calpain in Cngb1/Hcn1 double-KO mice. Finally, the deletion of HCN1 also exacerbated degeneration of cone photoreceptors in a mouse model of CNGA3-linked achromatopsia. Our results identify HCN1 as a major modifier of photoreceptor degeneration and suggest that pharmacological inhibition of HCN channels may enhance disease progression in RP and achromatopsia patients.

Retinitis pigmentosa: impact of different Pde6a point mutations on the disease phenotype.

Mutations in the PDE6A gene can cause rod photoreceptors degeneration and the blinding disease retinitis pigmentosa (RP). While a number of pathogenic PDE6A mutations have been described, little is known about their impact on compound heterozygous situations and potential interactions of different disease-causing alleles. Here, we used a novel mouse model for the Pde6a R562W mutation in combination with an existing line carrying the V685M mutation to generate compound heterozygous Pde6a V685M/R562W animals, exactly homologous to a case of human RP. We compared the progression of photoreceptor degeneration in these compound heterozygous mice with the homozygous V685M and R562W mutants, and additionally with the D670G line that is known for a relatively mild phenotype. We investigated PDE6A expression, cyclic guanosine mono-phosphate accumulation, calpain and caspase activity, in vivo retinal function and morphology, as well as photoreceptor cell death and survival. This analysis confirms the severity of different Pde6a mutations and indicates that compound heterozygous mutants behave like intermediates of the respective homozygous situations. Specifically, the severity of the four different Pde6a situations may be categorized by the pace of photoreceptor degeneration: V685M (fastest) > V685M/R562W > R562W > D670G (slowest). While calpain activity was strongly increased in all four mutants, caspase activity was not. This points to the execution of non-apoptotic cell death and may lead to the identification of new targets for therapeutic interventions. For individual RP patients, our study may help to predict time-courses for Pde6a-related retinal degeneration and thereby facilitate the definition of a window-of-opportunity for clinical interventions.

CRB2 acts as a modifying factor of CRB1-related retinal dystrophies in mice.

Mutations in the CRB1 gene lead to retinal dystrophies ranging from Leber congenital amaurosis (LCA) to early-onset retinitis pigmentosa (RP), due to developmental defects or loss of adhesion between photoreceptors and Müller glia cells, respectively. Whereas over 150 mutations have been found, no clear genotype-phenotype correlation has been established. Mouse Crb1 knockout retinas show a mild phenotype limited to the inferior quadrant, whereas Crb2 knockout retinas display a severe degeneration throughout the retina mimicking the phenotype observed in RP patients associated with CRB1 mutations. Crb1Crb2 double mutant retinas have severe developmental defects similar to the phenotype observed in LCA patients associated with CRB1 mutations. Therefore, CRB2 is a candidate modifying gene of human CRB1-related retinal dystrophy. In this study, we studied the cellular localization of CRB1 and CRB2 in human retina and tested the influence of the Crb2 gene allele on Crb1-retinal dystrophies in mice. We found that in contrast to mice, in the human retina CRB1 protein was expressed at the subapical region in photoreceptors and Müller glia cells, and CRB2 only in Müller glia cells. Genetic ablation of one allele of Crb2 in heterozygote Crb1(+/-) retinas induced a mild retinal phenotype, but in homozygote Crb1 knockout mice lead to an early and severe phenotype limited to the entire inferior retina. Our data provide mechanistic insight for CRB1-related LCA and RP.

Targeted ablation of Crb2 in photoreceptor cells induces retinitis pigmentosa.

In humans, the Crumbs homolog-1 (CRB1) gene is mutated in autosomal recessive Leber congenital amaurosis and early-onset retinitis pigmentosa. In mammals, the Crumbs family is composed of: CRB1, CRB2, CRB3A and CRB3B. Recently, we showed that removal of mouse Crb2 from retinal progenitor cells, and consequent removal from Müller glial and photoreceptor cells, results in severe and progressive retinal degeneration with concomitant loss of retinal function that mimics retinitis pigmentosa due to mutations in the CRB1 gene. Here, we studied the effects of cell-type-specific loss of CRB2 from the developing mouse retina using targeted conditional deletion of Crb2 in photoreceptors or Müller cells. We analyzed the consequences of targeted loss of CRB2 in the adult mouse retina using adeno-associated viral vectors encoding Cre recombinase and short hairpin RNA against Crb2. In vivo retinal imaging by means of optical coherence tomography on retinas lacking CRB2 in photoreceptors showed progressive thinning of the photoreceptor layer and cellular mislocalization. Electroretinogram recordings under scotopic conditions showed severe attenuation of the a-wave, confirming the degeneration of photoreceptors. Retinas lacking CRB2 in developing photoreceptors showed early onset of abnormal lamination, whereas retinas lacking CRB2 in developing Müller cells showed late onset retinal disorganization. Our data suggest that in the developing retina, CRB2 has redundant functions in Müller glial cells, while CRB2 has essential functions in photoreceptors. Our data suggest that short-term loss of CRB2 in adult mouse photoreceptors, but not in Müller glial cells, causes sporadic loss of adhesion between photoreceptors and Müller cells.

Mosaic synaptopathy and functional defects in Cav1.4 heterozygous mice and human carriers of CSNB2.

Mutations in CACNA1F encoding the α1-subunit of the retinal Cav1.4 L-type calcium channel have been linked to Cav1.4 channelopathies including incomplete congenital stationary night blindness type 2A (CSNB2), Åland Island eye disease (AIED) and cone-rod dystrophy type 3 (CORDX3). Since CACNA1F is located on the X chromosome, Cav1.4 channelopathies are typically affecting male patients via X-chromosomal recessive inheritance. Occasionally, clinical symptoms have been observed in female carriers, too. It is currently unknown how these mutations lead to symptoms in carriers and how the retinal network in these females is affected. To investigate these clinically important issues, we compared retinal phenotypes in Cav1.4-deficient and Cav1.4 heterozygous mice and in human female carrier patients. Heterozygous Cacna1f carrier mice have a retinal mosaic consistent with differential X-chromosomal inactivation, characterized by adjacent vertical columns of affected and non-affected wild-type-like retinal network. Vertical columns in heterozygous mice are well comparable to either the wild-type retinal network of normal mice or to the retina of homozygous mice. Affected retinal columns display pronounced rod and cone photoreceptor synaptopathy and cone degeneration. These changes lead to vastly impaired vision-guided navigation under dark and normal light conditions and reduced retinal electroretinography (ERG) responses in Cacna1f carrier mice. Similar abnormal ERG responses were found in five human CACNA1F carriers, four of which had novel mutations. In conclusion, our data on Cav1.4 deficient mice and human female carriers of mutations in CACNA1F are consistent with a phenotype of mosaic CSNB2.

Targeted ablation of CRB1 and CRB2 in retinal progenitor cells mimics Leber congenital amaurosis.

Development in the central nervous system is highly dependent on the regulation of the switch from progenitor cell proliferation to differentiation, but the molecular and cellular events controlling this process remain poorly understood. Here, we report that ablation of Crb1 and Crb2 genes results in severe impairment of retinal function, abnormal lamination and thickening of the retina mimicking human Leber congenital amaurosis due to loss of CRB1 function. We show that the levels of CRB1 and CRB2 proteins are crucial for mouse retinal development, as they restrain the proliferation of retinal progenitor cells. The lack of these apical proteins results in altered cell cycle progression and increased number of mitotic cells leading to an increased number of late-born cell types such as rod photoreceptors, bipolar and Müller glia cells in postmitotic retinas. Loss of CRB1 and CRB2 in the retina results in dysregulation of target genes for the Notch1 and YAP/Hippo signaling pathways and increased levels of P120-catenin. Loss of CRB1 and CRB2 result in altered progenitor cell cycle distribution with a decrease in number of late progenitors in G1 and an increase in S and G2/M phase. These findings suggest that CRB1 and CRB2 suppress late progenitor pool expansion by regulating multiple proliferative signaling pathways.

Loss of CRB2 in the mouse retina mimics human retinitis pigmentosa due to mutations in the CRB1 gene.

In humans, the Crumbs homolog-1 (CRB1) gene is mutated in progressive types of autosomal recessive retinitis pigmentosa and Leber congenital amaurosis. However, there is no clear genotype-phenotype correlation for CRB1 mutations, which suggests that other components of the CRB complex may influence the severity of retinal disease. Therefore, to understand the physiological role of the Crumbs complex proteins, we generated and analysed conditional knockout mice lacking CRB2 in the developing retina. Progressive disorganization was detected during late retinal development. Progressive thinning of the photoreceptor layer and sites of cellular mislocalization was detected throughout the CRB2-deficient retina by confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography. Under scotopic conditions using electroretinography, the attenuation of the a-wave was relatively stronger than that of the b-wave, suggesting progressive degeneration of photoreceptors in adult animals. Histological analysis of newborn mice showed abnormal lamination of immature rod photoreceptors and disruption of adherens junctions between photoreceptors, Müller glia and progenitor cells. The number of late-born progenitor cells, rod photoreceptors and Müller glia cells was increased, concomitant with programmed cell death of rod photoreceptors. The data suggest an essential role for CRB2 in proper lamination of the photoreceptor layer and suppression of proliferation of late-born retinal progenitor cells.

Gene therapy restores vision and delays degeneration in the CNGB1(-/-) mouse model of retinitis pigmentosa.

Retinitis pigmentosa (RP) is a group of genetically heterogeneous, severe retinal diseases commonly leading to legal blindness. Mutations in the CNGB1a subunit of the rod cyclic nucleotide-gated (CNG) channel have been found to cause RP in patients. Here, we demonstrate the efficacy of gene therapy as a potential treatment for RP by means of recombinant adeno-associated viral (AAV) vectors in the CNGB1 knockout (CNGB1(-/-)) mouse model. To enable efficient packaging and rod-specific expression of the relatively large CNGB1a cDNA (~4 kb), we used an AAV expression cassette with a short rod-specific promoter and short regulatory elements. After injection of therapeutic AAVs into the subretinal space of 2-week-old CNGB1(-/-) mice, we assessed the restoration of the visual system by analyzing (i) CNG channel expression and localization, (ii) retinal function and morphology and (iii) vision-guided behavior. We found that the treatment not only led to expression of full-length CNGB1a, but also restored normal levels of the previously degraded CNGA1 subunit of the rod CNG channel. Both proteins co-localized in rod outer segments and formed regular CNG channel complexes within the treated area of the CNGB1(-/-) retina, leading to significant morphological preservation and a delay of retinal degeneration. In the electroretinographic analysis, we also observed restoration of rod-driven light responses. Finally, treated CNGB1(-/-) mice performed significantly better than untreated mice in a rod-dependent vision-guided behavior test. In summary, this work provides a proof-of-concept for the treatment of rod channelopathy-associated RP by AAV-mediated gene replacement.

Gene therapy restores vision and delays degeneration in the CNGB1(-/-) mouse model of retinitis pigmentosa.

Retinitis pigmentosa (RP) is a severe retinal disease characterized by a progressive degeneration of rod photoreceptors and a secondary loss of cone function. Here, we used CNGB1-deficient (CNGB1(-/-)) mice, a mouse model for autosomal recessive RP, to evaluate the efficacy of adeno-associated virus (AAV) vector-mediated gene therapy for the treatment of RP. The treatment restored normal expression of rod CNG channels and rod-driven light responses in the CNGB1(-/-) retina. This led to a substantial delay of retinal degeneration and long-term preservation of retinal morphology. Finally, treated CNGB1(-/-) mice performed significantly better than untreated mice in a rod-dependent vision-guided behavior test. In summary, this study holds promise for the treatment of rod channelopathy-associated retinitis pigmentosa by AAV-mediated gene replacement.

Simultaneous exfoliation and functionalization of MoS2 with tetrapyridyl porphyrin.

Molybdenum disulfide (MoS2) attracts the attention of the scientific community due to its thickness dependent properties. To fully exploit these features, it is necessary to produce the material in mono or few-layers on a large scale. Several methodologies have been developed for this purpose, the most promising one being liquid phase exfoliation (LPE). LPE allows obtaining good quality exfoliated MoS2 in a simple and scalable manner. Herein we report the simultaneous exfoliation and functionalization of MoS2 in chloroform using a specific porphyrin, namely tetrapyridyl porphyrin. We have corroborated that the exfoliation of MoS2 in the volatile solvent increases in the presence of the porphyrin due to the different interactions between them, obtaining dispersions with good concentrations. Additionally, the optical properties of the porphyrin are modified by these interactions. The characterization carried out by several techniques supports the hypothesis that the interactions occur through the pyridyl rings of the porphyrin and the molybdenum atoms of the material.

Characterization of emerging 2D materials after chemical functionalization.

The chemical modification of 2D materials has proven a powerful tool to fine tune their properties. With this motivation, the development of new reactions has moved extremely fast. The need for speed, together with the intrinsic heterogeneity of the samples, has sometimes led to permissiveness in the purification and characterization protocols. In this review, we present the main tools available for the chemical characterization of functionalized 2D materials, and the information that can be derived from each of them. We then describe examples of chemical modification of 2D materials other than graphene, focusing on the chemical description of the products. We have intentionally selected examples where an above-average characterization effort has been carried out, yet we find some cases where further information would have been welcome. Our aim is to bring together the toolbox of techniques and practical examples on how to use them, to serve as guidelines for the full characterization of covalently modified 2D materials.

A "signal off-on" fluorescence bioassay based on 2D-MoS2-tetrahedral DNA bioconjugate for rapid virus detection.

In this work we present the preparation of a 2D molybdenum disulphide nanosheets (2D-MoS2) and tetrahedral DNA nanostructures (TDNs) bioconjugate, and its application to the development of a bioassay for rapid and easy virus detection. The bioconjugate has been prepared by using TDNs carrying the capture probe labelled with 6-carboxyfluoresceine (6-FAM). As case of study to assess the utility of the assay developed, we have chosen the SARS-CoV-2 virus. Hence, as probe we have used a DNA sequence complementary to a region of the SARS-CoV-2 ORF1ab gene (TDN-ORF-FAM). This 6-FAM labelled capture probe is located on the top vertex of the tetrahedral DNA nanostructure, the three left vertices of TDNs have a thiol group. These TDNs are bounded to 2D-MoS2 surface through the three thiol groups, allowing the capture probe to be oriented to favour the biorecognition reaction with the analyte. This biorecognition resulting platform has finally been challenged to the detection of the SARS-CoV-2 ORF1ab gene sequence as the target model by measuring fluorescence before and after the hybridization event with a detection limit of 19.7fM. Furthermore, due to high sensitivity of the proposed methodology, it has been applied to directly detect the virus in nasopharyngeal samples of infected patients without the need of any amplification step. The developed bioassay has a wide range of applicability since it can be applied to the detection of any pathogen by changing the probe corresponding to the target sequence. Thus, a novel, hands-on strategy for rapid pathogen detection has proposed and has a high potential application value in the early diagnosis of infections causes by virus or bacteria.

Rapid and simple viral protein detection by functionalized 2D MoS2/graphene electrochemiluminescence aptasensor.

In this work we present the development of an electrochemiluminescence aptasensor based on electrografting molybdenum disulphide nanosheets functionalized with diazonium salt (MoS2-N2+) upon screen-printed electrodes of graphene (SPEs GPH) for viral proteins detection. In brief, this aptasensor consists of SPEs GPH electrografted with MoS2-N2+ and modified with a thiolated aptamer, which can specifically recognize the target protein analyte. In this case, we have used SARS-CoV-2 spike protein as model protein. Electrochemiluminescence detection was performed by using the [Ru(bpy)3]2+/TPRA (tripropylamine) system, which allows the specific detection of the SARS-CoV-2 spike protein easily and rapidly with a detection limit of 9.74 fg/mL and a linear range from 32.5 fg/mL to 50.0 pg/mL. Moreover, the applicability of the aptasensor has been confirmed by the detection of the protein directly in human saliva samples. Comparing our device with a traditional saliva antigen test, our aptasensor can detect the spike protein even when the saliva antigen test gives a negative result.