Idiosyncatic recognition of Zn2+ and CN- using pyrazolyl-hydroxy-coumarin scaffold and live cell imaging: depiction of luminescent Zn(II)-metallocryptand.

Multi-responsive and selective sensor design is one of the stimulating research areas in the sensors field. We have designed a pyrazolyl-hydroxy-coumarin scaffold, 7-hydroxy-4-methyl-8-(((5-phenyl-1H-pyrazol-3-yl)imino)methyl)-2H-chromen-2-one (H2L) and characterized it by spectroscopic techniques (1H NMR, 13C NMR, ESI-MS, IR). The single crystal X-ray diffraction measurement confirms the molecular structure of the probe. It shows the selective sensing of Zn2+ in the presence of sixteen other cations with 'Turn On' approach through the enhancement of green florescence ((λem = 499 nm; λex = 390 nm) in CH3CN/H2O (99 : 1, v/v; HEPES buffer, pH 7.5) medium with the limit of detection (LOD) of 34.76 nM. The structural depiction of the isolated Zn2+ complex reveals cage like metallocryptand cyclic hexamer, [Zn6L6] with 30.9% void of cavity along the crystallographic c axis of approximate dimension of 7.502 × 7.050 × 7.068 Å3. The diffusion NMR study reveals only one type of complex in the solution, having 1 : 1 composition, i.e., Zn2+ : H2L, which affirms the isolated form of the complex. On the other hand, the receptor, H2L, recognizes the very noxious anion CN- out of sixteen anions. The product identification using spectroscopic techniques supports the nucleophilic addition of CN- across the exocyclic imine (CN) bond, which shows blue emission ((λem = 447 nm; λex = 390 nm), and the LOD was 19.91 nM. The composition of [H2L-Zn2+] and [H2L-CN-] was established by 1H NMR titration, Job's method, ESI-MS, and FTIR spectra. The efficacy of the probe was further studied using MTT assay in MDA-MB 231 and WI-38 cell line as well as for the intracellular imaging of Zn2+ and CN- using a fluorescence microscope. Flow Cytometry was further performed for the quantitative analysis of Zn2+ distribution in MDA-MB 231 cells.

Two-Dimensional MOF Modulated Fiber Nanogenerator for Effective Acoustoelectric Conversion and Human Motion Detection.

The real-time application of piezoelectric nanogenerators (PNGs) under a harsh environment remains a challenge due to lower output performance and poor durability. Thus, the development of flexible, sensitive, and stable PNGs became a topic of interest to capture different human motions including gesture monitoring to speech recognition. Herein, a scalable approach is adapted where naphthylamine bridging a [Cd(II)-µ-I4] two-dimensional (2D) metal-organic framework (MOF)-reinforced poly(vinylidene fluoride) (PVDF) composite nanofibers mat is prepared to fabricate a flexible and sensitive composite piezoelectric nanogenerator (C-PNG). The needle-shaped MOF was successfully synthesized by the layering and diffusion of two different solutions. The incorporation of single-crystalline 2D MOF ensures a large content of electroactive phases (98%) with a resultant high-magnitude piezoelectric coefficient of 41 pC/N in a composite nanofibers mat due to the interfacial specific interaction with -CH2-/-CF2- dipoles of PVDF. As an outcome, C-PNG generates high electrical output (open-circuit voltage of 22 V and maximum power density of 24 µW/cm2) with a very fast response time (tr ≈ 5 ms) under periodic pressure imparting stimuli. Benefiting from bending and twisting functionality, C-PNG is capable of scavenging biomechanical energy by mimicking complex musculoskeletal motions that broaden its application in wearable electronics and fabric integrated medical devices. In addition, C-PNG also demonstrates an efficient acoustic vibration to electric energy conversion capability with an improved power density and acoustic sensitivity of 6.25 µW and 0.95 V/Pa, respectively. The overall energy conversion efficiency is sufficient to operate several consumer electronics without any energy storage unit. This acoustic observation is further validated by the finite element method-based theoretical simulation. Overall, the 2D MOF-based device design strategy opens up a new possibility to develop a human-motion compatible energy generator and a self-powered acoustic sensor to power up electronic gadgets as well as low-frequency noise detection.

Reference standards for accurate validation and optimization of assays that determine integrated lentiviral vector copy number in transduced cells.

Lentiviral vectors (LV) have emerged as a robust technology for therapeutic gene delivery into human cells as advanced medicinal products. As these products are increasingly commercialized, there are concomitant demands for their characterization to ensure safety, efficacy and consistency. Standards are essential for accurately measuring parameters for such product characterization. A critical parameter is the vector copy number (VCN) which measures the genetic dose of a transgene present in gene-modified cells. Here we describe a set of clonal Jurkat cell lines with defined copy numbers of a reference lentiviral vector integrated into their genomes. Genomic DNA was characterized for copy number, genomic integrity and integration coordinates and showed uniform performance across independent quantitative PCR assays. Stability studies during continuous long-term culture demonstrated sustained renewability of the reference standard source material. DNA from the Jurkat VCN standards would be useful for control of quantitative PCR assays for VCN determination in LV gene-modified cellular products and clinical samples.

Three-Dimensional MOF-Assisted Self-Polarized Ferroelectret: An Effective Autopowered Remote Healthcare Monitoring Approach.

In recent years, flexible and sensitive pressure sensors are of extensive interest in healthcare monitoring, artificial intelligence, and national security. In this context, we report the synthetic procedure of a three-dimensional (3D) metal-organic framework (MOF) comprising cadmium (Cd) metals as nodes and isoniazid (INH) moieties as organic linkers (CdI2-INH═CMe2) for designing self-polarized ferroelectret-based highly mechano-sensitive skin sensors. The as-synthesized MOF preferentially nucleates the stable piezoelectric ß-phase in poly(vinylidene fluoride) (PVDF) and also gives rise to a porous ferroelectret composite film. Benefiting from the porous structure of 3D MOFs, composite ferroelectret film-based ultrasensitive pressure sensor (mechano-sensitivity of 8.52 V/kPa within 1 kPa pressure range) as well as high-throughput ( power density of 32 µW/cm2) mechanical energy harvester (MEH) has been designed. Simulation-based finite element method (FEM) analysis indicates that the geometrical stress confinement effect within the interpore region of the ferroelectret structure synergistically influences the mechano-electrical property of the MEH. In addition, 143 pC/N (∼4.5 times higher than commercial piezoelectric PVDF films) piezoelectric charge coefficient (d33) magnitude and superior response time (tr ∼ 8 ms) of this composite ferroelectret film enable the detection of different physiological signals such as coughing, pronunciation, and gulping behavior, making it a promising candidate for early intervention of healthcare, which may play a significant role in accurate alert of influenza and chronic obstructive pulmonary disease (COPD)-related symptoms. In addition, MEH enables the tracking of the subtle pressure change in the wrist pulse, indicating its usefulness in effective mechano-sensitivity. Since the cardiovascular signal is one of the vital parameters that can determine the on-going physiological conditions, the wireless transmission of the detected wrist pulse signal has been demonstrated. All of these features coupled with wireless data transmission indicate the promising application of MOF-assisted composite ferroelectret films in noninvasive real-time remote healthcare monitoring.

Studies on Magnetic and Dielectric Properties of Antiferromagnetically Coupled Dinuclear Cu(II) in a One-Dimensional Cu(II) Coordination Polymer.

A one-dimensional Cu(II) coordination polymer with encapsulated antiferromagnetically coupled binuclear Cu(II) has been synthesized by using 5-nitroisophthalic acid (5-N-IPA) and 4-aminopyridine (4-APY) [Cu2(5-N-IPA)2(4-APY)4] n (1). Electrical properties are examined by complex impedance (Z*), dielectric permittivity (ε*), and ac conductivity studies at different frequencies (10 kHz-5 MHz) and temperatures (253-333 K). The contribution of grain and grain boundary has been explained by a different theoretical model. The variable temperature magnetic susceptibility data for compound 1 were recorded between 300 and 2 K. The shape of the curve (χM T vs T) indicates dominant antiferromagnetic coupling, which results from the interaction between the copper(II) atoms.

Three-Dimensional-Coordination Polymer of Zn(II)-Carboxylate: Structural Elucidation, Photoelectrical Conductivity, and Biological Activity.

A newly designed mixed-ligand coordination polymer [Zn4(bdc)4(ppmh)2(H2O)] n (1) (H2 bdc = 1,4-benzene dicarboxylic acid, ppmh = N-pyridin-2-yl-N'-pyridin-4-ylmethylene-hydrazine) has been characterized using different physicochemical techniques. The structure has been confirmed by single crystal X-ray diffraction measurements. There are two pyridyl-N and one hydrazino-imine-N donor centers in ppmh, where two pyridyl-Ns bind simultaneously to two Zn(II) to serve as a bridging agent to form a coordination polymer. The 1,4-benzene dicarboxylato (bdc) is ligated via the aromatic dicarboxylato-O to form a one-dimensional (1D) chain. These two 1D chains about Zn(II) constitute a two-dimensional structure, which undergoes noncovalent interactions (C-H···π and π···π) to generate a three-dimensional supramolecular assembly. Electrical conductivity of 1 is higher by 1 order (1.37 × 10-6 S/cm) than that of the free ligand, ppmh (6.2 × 10-7 S/cm). Especially, the responsivity of the compound 1 was 56.21 mA/W, which is 11 times higher than that of the ligand ppmh (5.12 mA/W). The specific detectivity of the compound was 2.17 × 1010 Jones, which is also almost 10 times higher with respect to the specific detectivity of the ligand-based device (4.53 × 109 Jones). The results show that the compound can be valuable for optoelectronic fields. The biological studies suggest that compound 1 is antibacterial as well as a promising anticancer agent (LD50, 42.2 µg/mL against HepG2 cells), while ligands remain silent. Investigation of the mechanism of the cell killing activity of compound 1 accounts the generation of intracellular reactive oxygen species.

The type of responder T-cell has a significant impact in a human in vitro suppression assay.

BACKGROUND: In type 1 diabetes (T1D), a prototypic autoimmune disease, effector T cells destroy beta cells. Normally, CD4(+)CD25(+high), or natural regulatory T cells (Tregs), counter this assault. In autoimmunity, the failure to suppress CD4(+)CD25(low) T cells is important for disease development. However, both Treg dysfunction and hyperactive responder T-cell proliferation contribute to disease. METHODS/PRINCIPAL FINDINGS: We investigated human CD4(+)CD25(low) T cells and compared them to CD4(+)CD25(-) T cells in otherwise equivalent in vitro proliferative conditions. We then asked whether these differences in suppression are exacerbated in T1D. In both single and co-culture with Tregs, the CD4(+)CD25(low) T cells divided more rapidly than CD4(+)CD25(-) T cells, which manifests as increased proliferation/reduced suppression. Time-course experiments showed that this difference could be explained by higher IL-2 production from CD4+CD25(low) compared to CD4+CD25- T cells. There was also a significant increase in CD4+CD25(low) T-cell proliferation compared to CD4+CD25- T cells during suppression assays from RO T1D and at-risk subjects (n = 28, p = 0.015 and p = 0.024 respectively). CONCLUSIONS/SIGNIFICANCE: The in vitro dual suppression assays proposed here could highlight the impaired sensitivity of certain responder T cells to the suppressive effect of Tregs in human autoimmune diseases.

Inducible regulatory T cells (iTregs) from recent-onset type 1 diabetes subjects show increased in vitro suppression and higher ITCH levels compared with controls.

CD4+CD25+(high) regulatory T cells (Tregs) play a pivotal role in the control of the immune response. A growing body of evidence suggests the reduced function of these cells in autoimmune diseases, including type 1 diabetes (T1D). Restoration of their function can potentially delay further disease development. In the present study, we have converted conventional effector T cells into induced Tregs (iTregs) in recent-onset (RO) T1D (n=9) and compared them with the same cells generated in controls (n=12) and in long-standing (LS) T1D subjects (n=9). The functional potential of in-vitro-generated Tregs was measured by using an in vitro proliferation assay. We noted that the suppressive potential of iTregs exceeded that of natural regulatory T cells (nTregs) only in the RO T1D subjects. We showed that iTregs from RO T1D subjects had increased expression of Foxp3, E3 ubiquitin ligase (ITCH) and TGF-beta-inducible early gene 1 (TIEG1) compared with control and LS T1D subjects. We also expanded natural, thymically derived Tregs (nTregs) and compared the functional ability of these cells between subject groups. Expanded cells from all three subject groups were suppressive. RO T1D subjects were the only group in which both iTregs and expanded Tregs were functional, suggesting that the inflammatory milieu impacts in vitro Treg generation. Future longitudinal studies should delineate the actual contribution of the stage of disease to the quality of in-vitro-generated Tregs.

The role of NF-kappaB and Smad3 in TGF-beta-mediated Foxp3 expression.

The transcription factor Foxp3 is essential for the development of functional, natural Treg (nTreg), which plays a prominent role in self-tolerance. Suppressive Foxp3(+) Treg cells can be generated from naïve T cells ex vivo, following TCR and TGF-beta1 stimulations. However, the molecular contributions from the different arms of these pathways leading to Foxp3 expression are not fully understood. TGF-beta1-activated Smad3 plays a major role in the expression of Foxp3, since TGF-beta1-induced-Treg generation from Smad3(-/-) mice is markedly reduced and abolished by inactivating Smad2. In the TCR pathway, deletion of Bcl10, which activates NF-kappaB, markedly reduces both IL-2 and Foxp3 production. However, partial rescue of Foxp3 expression occurs on addition of exogenous IL-2. TGF-beta1 significantly attenuates NF-kappaB binding to the Foxp3 promoter, while inducing Foxp3 expression. Furthermore, deletion of p50, a NF-kappaB subunit, results in increased Foxp3 expression despite a decline in the IL-2 production. We posit several TCR-NF-kappaB pathways, some increasing (Bcl10-IL-2-Foxp3) while others decreasing (p50-Foxp3) Foxp3 expression, with the former predominating. A better understanding of Foxp3 regulation could be useful in dissecting the cause of Treg dysfunction in several autoimmune diseases and for generating more potent TGF-beta1-induced-Treg cells for therapeutic purposes.

Apoptosis of CD4+ CD25(high) T cells in type 1 diabetes may be partially mediated by IL-2 deprivation.

BACKGROUND: Type 1 diabetes (T1D) is a T-cell mediated autoimmune disease targeting the insulin-producing pancreatic beta cells. Naturally occurring FOXP3(+)CD4(+)CD25(high) regulatory T cells (T(regs)) play an important role in dominant tolerance, suppressing autoreactive CD4(+) effector T cell activity. Previously, in both recent-onset T1D patients and beta cell antibody-positive at-risk individuals, we observed increased apoptosis and decreased function of polyclonal T(regs) in the periphery. Our objective here was to elucidate the genes and signaling pathways triggering apoptosis in T(regs) from T1D subjects. PRINCIPAL FINDINGS: Gene expression profiles of unstimulated T(regs) from recent-onset T1D (n = 12) and healthy control subjects (n = 15) were generated. Statistical analysis was performed using a Bayesian approach that is highly efficient in determining differentially expressed genes with low number of replicate samples in each of the two phenotypic groups. Microarray analysis showed that several cytokine/chemokine receptor genes, HLA genes, GIMAP family genes and cell adhesion genes were downregulated in T(regs) from T1D subjects, relative to control subjects. Several downstream target genes of the AKT and p53 pathways were also upregulated in T1D subjects, relative to controls. Further, expression signatures and increased apoptosis in T(regs) from T1D subjects partially mirrored the response of healthy T(regs) under conditions of IL-2 deprivation. CD4(+) effector T-cells from T1D subjects showed a marked reduction in IL-2 secretion. This could indicate that prior to and during the onset of disease, T(regs) in T1D may be caught up in a relatively deficient cytokine milieu. CONCLUSIONS: In summary, expression signatures in T(regs) from T1D subjects reflect a cellular response that leads to increased sensitivity to apoptosis, partially due to cytokine deprivation. Further characterization of these signaling cascades should enable the detection of genes that can be targeted for restoring T(reg) function in subjects predisposed to T1D.

Impaired survival of peripheral T cells, disrupted NK/NKT cell development, and liver failure in mice lacking Gimap5.

The loss of Gimap5 (GTPase of the immune-associated protein 5) gene function is the underlying cause of lymphopenia and autoimmune diabetes in the BioBreeding (BB) rat. The in vivo function of murine gimap5 is largely unknown. We show that selective gene ablation of the mouse gimap5 gene impairs the final intrathymic maturation of CD8 and CD4 T cells and compromises the survival of postthymic CD4 and CD8 cells, replicating findings in the BB rat model. In addition, gimap5 deficiency imposes a block of natural killer (NK)- and NKT-cell differentiation. Development of NK/NKT cells is restored on transfer of gimap5(-/-) bone marrow into a wild-type environment. Mice lacking gimap5 have a median survival of 15 weeks, exhibit chronic hepatic hematopoiesis, and in later stages show pronounced hepatocyte apoptosis, leading to liver failure. This pathology persists in a Rag2-deficient background in the absence of mature B, T, or NK cells and cannot be adoptively transferred by transplanting gimap5(-/-) bone marrow into wild-type recipients. We conclude that mouse gimap5 is necessary for the survival of peripheral T cells, NK/NKT-cell development, and the maintenance of normal liver function. These functions involve cell-intrinsic as well as cell-extrinsic mechanisms.

At-risk and recent-onset type 1 diabetic subjects have increased apoptosis in the CD4+CD25+ T-cell fraction.

BACKGROUND: In experimental models, Type 1 diabetes T1D can be prevented by adoptive transfer of CD4+CD25+ (FoxP3+) suppressor or regulatory T cells. Recent studies have found a suppression defect of CD4+CD25+(high) T cells in human disease. In this study we measure apoptosis of CD4+CD25+(high) T cells to see if it could contribute to reduced suppressive activity of these cells. METHODS AND FINDINGS: T-cell apoptosis was evaluated in children and adolescent 35 females/40 males subjects comprising recent-onset and long-standing T1D subjects and their first-degree relatives, who are at variable risk to develop T1D. YOPRO1/7AAD and intracellular staining of the active form of caspase 3 were used to evaluate apoptosis. Isolated CD4+CD25+(high) and CD4+CD25- T cells were co-cultured in a suppression assay to assess the function of the former cells. We found that recent-onset T1D subjects show increased apoptosis of CD4+CD25+(high) T cells when compared to both control and long-standing T1D subjects p<0.0001 for both groups. Subjects at high risk for developing T1D 2-3Ab+ve show a similar trend p<0.02 and p<0.01, respectively. On the contrary, in long-standing T1D and T2D subjects, CD4+CD25+(high) T cell apoptosis is at the same level as in control subjects p = NS. Simultaneous intracellular staining of the active form of caspase 3 and FoxP3 confirmed recent-onset FoxP3+ve CD4+CD25+(high) T cells committed to apoptosis at a higher percentage 15.3+/-2.2 compared to FoxP3+ve CD4+CD25+(high) T cells in control subjects 6.1+/-1.7 p<0.002. Compared to control subjects, both recent-onset T1D and high at-risk subjects had significantly decreased function of CD4+CD25+(high) T cells p = 0.0007 and p = 0.007, respectively. CONCLUSIONS: There is a higher level of ongoing apoptosis in CD4+CD25+(high) T cells in recent-onset T1D subjects and in subjects at high risk for the disease. This high level of CD4+CD25+(high) T-cell apoptosis could be a contributing factor to markedly decreased suppressive potential of these cells in recent-onset T1D subjects.