Recent Developments and Applications of Microbial Electrochemical Biosensors.

This chapter provides a comprehensive overview of microbial electrochemical biosensors, which are a unique class of biosensors that utilize the metabolic activity of microorganisms to convert chemical signals into electrical signals. The principles and mechanisms of these biosensors are discussed, including the different types of microorganisms that can be used. The various applications of microbial electrochemical biosensors in fields such as environmental monitoring, medical diagnostics, and food safety are also explored. The chapter concludes with a discussion of future research directions and potential advancements in the field of microbial electrochemical biosensors.

Exploitation of a 1D coordination polymer as a portable kit for an eye-catching fluorometric response towards sensing of trivalent cations.

The development and utilization of coordination polymers (CPs) have drawn interest for potential applications in different fields. Detection of metal ions in efficient and selective manners is an important field of research. It paves the way to protect human health by balancing toxic metal ions and biologically active metal ions in the atmosphere. In this regard, a new one-dimensional (1D) 4-(1-naphthylvinyl)pyridine (4-nvp) based CP [Cd(NCS)2(4-nvp)2]n (1) was synthesized and characterized structurally by single-crystal X-ray diffraction. Interestingly, this 1D CP underwent supramolecular aggregation via π⋯π stacking interactions, which specifically generated an environment for a potent "turn on" response in the presence of trivalent cations (Fe3+, Al3+, and Cr3+) in the nanomolar range but remained silent in the presence of other metal ions. Density functional theory (DFT) computations and X-ray photoelectron spectroscopy (XPS) were performed to establish the sensing phenomena. Fascinatingly, utilizing the sensitivity of 1 in an aqueous medium, a hands-on portable cotton swab kit was developed for instant identification of these three important trivalent metal cations.

The biophysical property of the limbal niche maintains stemness through YAP.

The cell fate decisions of stem cells (SCs) largely depend on signals from their microenvironment (niche). However, very little is known about how biochemical niche cues control cell behavior in vivo. To address this question, we focused on the corneal epithelial SC model in which the SC niche, known as the limbus, is spatially segregated from the differentiation compartment. We report that the unique biomechanical property of the limbus supports the nuclear localization and function of Yes-associated protein (YAP), a putative mediator of the mechanotransduction pathway. Perturbation of tissue stiffness or YAP activity affects SC function as well as tissue integrity under homeostasis and significantly inhibited the regeneration of the SC population following SC depletion. In vitro experiments revealed that substrates with the rigidity of the corneal differentiation compartment inhibit nuclear YAP localization and induce differentiation, a mechanism that is mediated by the TGFß-SMAD2/3 pathway. Taken together, these results indicate that SC sense biomechanical niche signals and that manipulation of mechano-sensory machinery or its downstream biochemical output may bear fruits in SC expansion for regenerative therapy.

A Gelation-Induced Enhanced Emission Active Stimuli Responsive and Superhydrophobic Organogelator: "Turn-On" Fluorogenic Detection of Cyanide and Dual-Channel Sensing of Nitroexplosives on Multiple Platforms.

A pyrene-based highly emissive low-molecular-weight organogelator, [2-(4-fluorophenyl)-3-(pyren-1-yl)acrylonitrile] (F1), is presented, which divulges thixotropic and thermochromic fluorescence switching via reversible gel-to-sol transition and tremendous superhydrophobicity (mean contact angles: 149-160°), devoid of any gelling and/or hydrophobic units. The rationale for the design strategy reveals that the restricted intramolecular rotation (RIR) in J-type self-assembly promotes F1 for the prolific effects of aggregation- and gelation-induced enhanced emission (AIEE and GIEE). Meanwhile, hindrance in charge transfer via the nucleophilic reaction of cyanide (CN-) on the C═C unit in F1 facilitates the selective fluorescence "turn-on" response in both solution [9:1 (v/v) DMSO/water] and solid state [paper kits] with significantly lower detection limits (DLs) of 37.23 nM and 13.4 pg/cm2, respectively. Subsequently, F1 discloses CN- modulated colorimetric and fluorescence "turn-off" dual-channel response for aqueous 2,4,6-trinitrophenol (PA) and 2,4-dinitrophenol (DNP) in both solution (DL = 49.98 and 44.1 nM) and solid state (DL = 114.5 and 92.05 fg/cm2). Furthermore, the fluorescent nanoaggregates of F1 in water and its xerogel films permit a rapid dual-channel "on-site" detection of PA and DNP, where the DLs ranged from nanomolar (nM) to sub-femtogram (fg) levels. Mechanistic insights reveal that the ground-state electron transfer from the fluorescent [F1-CN] ensemble to the analytes is responsible for anion driven sensory response, whereas the unusual inner filter effect (IFE) driven photoinduced electron transfer (PET) was responsible for self-assembled F1 response toward desired analytes. Additionally, the nanoaggregates and xerogel films also detect PA and DNP in their vapor phase with reasonable percentage of recovery from the soil and river water samples. Therefore, the elegant multifunctionality from a single luminogenic framework allows F1 to provide a smart pathway for achieving environmentally benign real-world applications on multiple platforms.

Ferric reductase-related proteins mediate fungal heme acquisition.

Heme can serve as iron source in many environments, including the iron-poor animal host environment. The fungal pathobiont Candida albicans expresses a family of extracellular CFEM hemophores that capture heme from host proteins and transfer it across the cell wall to the cell membrane, to be endocytosed and utilized as heme or iron source. Here, we identified Frp1 and Frp2, two ferric reductase (FRE)-related proteins that lack an extracellular N-terminal substrate-binding domain, as being required for hemoglobin heme utilization and for sensitivity to toxic heme analogs. Frp1 and Frp2 redistribute to the plasma membrane in the presence of hemin, consistent with a direct role in heme trafficking. Expression of Frp1 with the CFEM hemophore Pga7 can promote heme utilization in Saccharomyces cerevisiae as well, confirming the functional interaction between these proteins. Sequence and structure comparison reveals that the CFEM hemophores are related to the FRE substrate-binding domain that is missing in Frp1/2. We conclude that Frp1/2 and the CFEM hemophores form a functional complex that evolved from FREs to enable extracellular heme uptake.

Hosts and disease-causing fungi are often locked into a battle over resources. The host will attempt to withhold molecules that the fungus needs to survive, while the pathogen will try to find alternative routes to obtain them. Candida albicans, for example, can go after the atoms of iron embedded in the proteins of the organism it infects. To do so it releases molecules known as hemophores, which scavenge the iron-containing heme molecule that equips oxygen-carrying proteins in the blood. Once captured, the heme is carried across the wall that protects C. albicans from the environment and brought to the membrane of the cell. It is then taken in and trafficked inside vesicles to its destination. However, the identity of the molecular actors which help to bridge the internal and external segments of the heme journey remain unclear. Previous studies have shown that the hemophore Pga7 is involved, but this protein is attached to the outside of the cell membrane, where it cannot directly interact with the import machinery. Roy et al. set out to discover this missing link. Examining the genomes of fungal species related to C. albicans highlighted two membrane proteins, Frp1 and Frp2, which could participate in heme uptake. Protein sequence comparison revealed that Frp1 and Frp2 were closely related to ferric reductases, a group of membrane enzymes which can chemically alter extracellular iron prior to uptake. Deleting the genes for Frp1 and Frp2 rendered C. albicans cells incapable of taking in heme. Conversely, a fungal species which cannot normally uptake heme could efficiently internalise these complexes when artificially equipped with Frp1 and Pga7, suggesting that the two proteins work closely together. Finally, protein structure comparisons highlighted that an extracellular domain present in ferric reductases but absent in Frp1 and Frp2 is, in fact, related to Pga7 and other hemophores. This implies that the iron and heme uptake systems may share a common evolutionary origin. Overall, the work by Roy et al. reveals a new family of proteins which allow disease-causing fungi to steal iron from their hosts. This knowledge may be useful to design better anti-fungal treatments.

A highly emissive Zn(II)-pyridyl-benzimidazolyl-phenolato-based chemosensor: detection of H2PO4-via "use" and "throw" device fabrication.

2-Ethoxy-6-[1-(phenyl-pyridin-2-yl-methyl)-1H-benzoimidazol-2-yl]-phenol (HL) selectively serves as a sensitive 'turn on' Zn2+ sensor in 9 : 1 (v/v) DMSO/H2O (HEPES buffer, pH = 7.4) medium in the presence of sixteen other cations at the limit of detection (LOD) of 3.2 nM. The strong blue emission of the complex, {[Zn(L1)OAc]} (HL1 = benzimidazolyl ring-opening structure of HL) (λem, 461 nm), is quenched by H2PO4- in the presence of eighteen other anions and the LOD is 0.238 µM. The emission of the complex is due to restricted intramolecular rotation (RIR) followed by chelation-enhanced fluorescence (CHEF). The quenching of the emission of [Zn(L1)OAc] by H2PO4- (in the presence of other PVs (inorganic and biological) as well as additional anions) is due to the 'turn off' fluorescence via the demetallation and release of the nonfluorescent ligand, HL, and [Zn(H2PO4)]+. An INHIBIT logic gate memory circuit of the probe HL was devised with Zn2+ and H2PO4- as two consecutive inputs. The percentage of H2PO4- recovery was excellent and was obtained from distilled, tap, and drinking water sources. The bright blue emission of [Zn(L1)OAc] further triggered the fabrication of ready-made portable thin films of the Zn-complex, which executed a cost-effective 'on-site' solid-state contact mode detection of H2PO4- with selectivity at the picogram level (10.97 pg cm-2) by monitoring the intensities of quenched spots under UV light upon varying the analyte concentration from 10-8 to 10-3 M. Finally, taking advantage of reversible fluorescence switching, a simple and definite ion-responsive security feature was successfully embedded into a "use" and "throw" solution-coated paper strip of the Zn(II)-pyridyl-benzimidazolyl-phenolato-based chemosensor, which efficiently detected H2PO4- in water by a successive 'ON-OFF' fluorescence switching-driven security activity without any exhaustion of the emission phenomenon.

Discrete limbal epithelial stem cell populations mediate corneal homeostasis and wound healing.

The accessibility and transparency of the cornea permit robust stem cell labeling and in vivo cell fate mapping. Limbal epithelial stem cells (LSCs) that renew the cornea are traditionally viewed as rare, slow-cycling cells that follow deterministic rules dictating their self-renewal or differentiation. Here, we combined single-cell RNA sequencing and advanced quantitative lineage tracing for in-depth analysis of the murine limbal epithelium. These analysis revealed the co-existence of two LSC populations localized in separate and well-defined sub-compartments, termed the "outer" and "inner" limbus. The primitive population of quiescent outer LSCs participates in wound healing and boundary formation, and these cells are regulated by T cells, which serve as a niche. In contrast, the inner peri-corneal limbus hosts active LSCs that maintain corneal epithelial homeostasis. Quantitative analyses suggest that LSC populations are abundant, following stochastic rules and neutral drift dynamics. Together these results demonstrate that discrete LSC populations mediate corneal homeostasis and regeneration.

A single cell atlas of human cornea that defines its development, limbal progenitor cells and their interactions with the immune cells.

PURPOSE: Single cell (sc) analyses of key embryonic, fetal and adult stages were performed to generate a comprehensive single cell atlas of all the corneal and adjacent conjunctival cell types from development to adulthood. METHODS: Four human adult and seventeen embryonic and fetal corneas from 10 to 21 post conception week (PCW) specimens were dissociated to single cells and subjected to scRNA- and/or ATAC-Seq using the 10x Genomics platform. These were embedded using Uniform Manifold Approximation and Projection (UMAP) and clustered using Seurat graph-based clustering. Cluster identification was performed based on marker gene expression, bioinformatic data mining and immunofluorescence (IF) analysis. RNA interference, IF, colony forming efficiency and clonal assays were performed on cultured limbal epithelial cells (LECs). RESULTS: scRNA-Seq analysis of 21,343 cells from four adult human corneas and adjacent conjunctivas revealed the presence of 21 cell clusters, representing the progenitor and differentiated cells in all layers of cornea and conjunctiva as well as immune cells, melanocytes, fibroblasts, and blood/lymphatic vessels. A small cell cluster with high expression of limbal progenitor cell (LPC) markers was identified and shown via pseudotime analysis to give rise to five other cell types representing all the subtypes of differentiated limbal and corneal epithelial cells. A novel putative LPCs surface marker, GPHA2, expressed on the surface of 0.41% ± 0.21 of the cultured LECs, was identified, based on predominant expression in the limbal crypts of adult and developing cornea and RNAi validation in cultured LECs. Combining scRNA- and ATAC-Seq analyses, we identified multiple upstream regulators for LPCs and demonstrated a close interaction between the immune cells and limbal progenitor cells. RNA-Seq analysis indicated the loss of GPHA2 expression and acquisition of proliferative limbal basal epithelial cell markers during ex vivo LEC expansion, independently of the culture method used. Extending the single cell analyses to keratoconus, we were able to reveal activation of collagenase in the corneal stroma and a reduced pool of limbal suprabasal cells as two key changes underlying the disease phenotype. Single cell RNA-Seq of 89,897 cells obtained from embryonic and fetal cornea indicated that during development, the conjunctival epithelium is the first to be specified from the ocular surface epithelium, followed by the corneal epithelium and the establishment of LPCs, which predate the formation of limbal niche by a few weeks. CONCLUSIONS: Our scRNA-and ATAC-Seq data of developing and adult cornea in steady state and disease conditions provide a unique resource for defining genes/pathways that can lead to improvement in ex vivo LPCs expansion, stem cell differentiation methods and better understanding and treatment of ocular surface disorders.

X-ray structure of two Schiff bases: TURN-ON sensing of Fe3+ and Al3+ in the HepG2 cell line.

The efficiency of the fluorescence sensitivity of a sensor may be tuned by the modulation of the steric and electronic parameters in the structure. In this study, the thiophenyl Schiff base (E)-N1-(phenyl(pyridin-2-yl)methyl)-N2-(thiophen-2-ylmethylene)benzene-1,2-diamine (HL') exhibited very high selectivity and a sensitive fluorescence enhancement towards Fe3+ with violet emission (λem, 385 nm; LOD, 3.8 nM). On the other hand, the naphthyl Schiff base (E)-1-(((2-((phenyl(pyridin-2-yl)methyl)amino)phenyl)imino)methyl)naphthalen-2-ol (H2L'') exhibited fluorescence sensitivity towards Al3+, showing blue emission (λem, 502 nm; LOD, 3.3 nM) in H2O (HEPES buffer, pH 7.4) medium. The emission enhancement of HL' upon binding to Fe3+ may be considered to be due to the restriction of intramolecular rotation, while the selectivity of H2L'' towards Al3+ may be due to the turn on emission through the restriction of excited state intramolecular proton transfer (ESIPT) and the introduction of chelation enhanced fluorescence (CHEF). Furthermore, DFT computation supported the sensing strategy and the probes were applied for intracellular detection of Fe3+ and Al3+ in HepG2 cell lines.

Chromogenic hydrazide Schiff base reagent: Spectrophotometric determination of CN- ion.

A Schiff base reagent, Picolinohydrazide-naphthol (HL), is used for trace level detection of toxic CN- selectively in presence of eighteen other anions (SCN-, OCN-, S2O32-, HPO42-, H2PO4-, I-, ClO4-, HSO4-, SO42-, AsO43-, NO2-, AsO2-, Cl-, F-, HF2-, NO3-, Br-, N3-) by visual color change, colorless to yellow, in DMSO/H2O (9:1, v/v) at pH, 7.2 (HEPES buffer) medium. The sensitivity of the probe shows that the limit of detection (LOD) is 7.08 µM. The probable mechanism for the sensing behavior involves the deprotonation of naphthol-OH by CN- that has been authenticated by 1H NMR titration and Mass spectra. The composition (1:1 mol ratio) is supported by Job's plot and binding constant (Ka, 1.5 × 104 M-1) is reported by Benesi-Hildebrand plot. Furthermore, a simple paper strip device is fabricated for the determination of CN- ion in water. DFT computation is carried out to explain the electronic spectral feature of the sensor.

Designing of Pb(II)-Based Novel Coordination Polymers (CPs): Structural Elucidation and Optoelectronic Application.

[Pb2(bdc)1.5(aiz)] n (1) and [Pb2(bdc)1.5(aiz)(MeOH)2] n (2) (H2bdc = 1,4-benzene dicarboxylic acid, aiz = (E)-N'-(thiophen-2-ylmethylene)isonicotinohydrazide) have been synthesized, and structural characterization has been established by X-ray analysis and thermogravimetric analysis (TGA). Here, bdc2- links two Pb(II) centers and the aiz ligand binds the metal centers in two different manners: chelating and monodonating. Thus, polymerizations have taken place from the combination of mixed ligand system. Optical band gaps have been studied via UV measurements. Again, the experimental and calculated (from density functional theory (DFT)) band gaps agree well and the semiconducting properties of synthesized polymeric materials have been approved. Thus, optoelectronic and photonic devices can be made by this type of coordination polymers (CPs). The I-V representative curves of 1 (device-A) and 2 (device-B) in both dark and illuminated conditions show that device-A has a higher magnitude of current than device-B. Dark- and photo-conductivity values of device-A are calculated as 2.94 × 10-6 and 6.12 × 10-6 S m-1, respectively, whereas for device-B, the values of dark- and photo-conductivity are 2.92 × 10-7 and 3.66 × 10-7 S m-1, respectively, at room temperature.

The oxidative dehydrogenation of a coumarinyl scaffold with copper ion and metal ion detection in human liver cancer cells (HepG2).

An unsymmetrical o-phenylenediamine derivative, L (7-hydroxy-4-methyl-8-(1-(phenyl- (pyridin-2-yl)methyl)-1H-benzo[d]imidazol-2-yl)-2H-chromen-2-one), has been synthesized from (E)-N1-(phenyl(pyridine-2-yl)methylene)benzene-1,2-diamine with 7-hydroxy-4-methyl-2-oxo-2H-chromene-8-carbaldehyde and characterized by X-ray, IR, 1H NMR and ESI-MS spectral analyses. The X-ray structure shows L as a cyclic benzimidazole derivative, but it undergoes ring-opening upon reaction with transition metal ions. L is non-emissive in 9 : 1 (v/v) EtOH/H2O (HEPES buffer, pH 7.4) but becomes highly fluorescent upon Zn2+ coordination, and the emissive Zn(ii) complex undergoes transmetallation in the presence of Cu2+ ions specifically, followed by amine to imine oxidation, i.e. an oxidative dehydrogenation (OD) reaction -(2e + 2H+) occurs. The transmetallation of Zn2+ from the complex by Cu2+ (CuCl2) separated the non-emissive X-ray diffractable crystal of [Cu(L'')Cl] (L'' = amine oxidized form of L). A square pyramidal [Cu(L'')][ClO4] complex was also isolated from the reaction of L with Cu(CH3CN)4(ClO4) in the presence of air, and in this complex the amine is also oxidized to the imine. Here, copper ions in the presence of air play an important role in the OD reaction of L as determined by EPR and cyclic voltammetry studies. The ligand, L, is used for Zn2+ ion recovery from a municipally supplied water sample. A paper strip detection kit for Zn2+ and Cu2+ is designed using L. The ligand is also used for intracellular Zn2+ detection in a human liver cancer cell line (HepG2).

Aggregation-Induced Emission-Active Hydrazide-Based Probe: Selective Sensing of Al3+, HF2 -, and Nitro Explosives.

(E)-N'-((2-Hydroxynaphthalen-1-yl)methylene)picolinohydrazide (H-PNAP) shows aggregation-induced emission (AIE) strictly in a 90% water/MeOH (v/v) mixture at 540 nm, and the solid-state emission is blue-shifted to 509 nm upon excitation at 400 nm. The AIE activity of H-PNAP is selectively quenched by 2,4,6-trinitrophenol (TNP) and 2,4-dinitrophenol (DNP) out of different nitroaromatic compounds with a limit of detection (LOD) of 7.79 × 10-7 and 9.08 × 10-7 M, respectively. The probe is nonemissive in aqueous medium (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, HEPES buffer, pH 7.2); however, it shows a strong emission to Al3+ (λem, 490 nm) in the presence of 17 other biological metal ions, and the LOD is 2.09 nM which is far below the WHO recommended value (7.41 mM). The emission of the [Al(PNAP)(NO3)2] complex is quenched by HF2 - (F- and PO4 3- are weak quencher), and the LOD is as low as 15 nM. The probable mechanism of the sensing feature of the probe has been authenticated by 1H nuclear magnetic resonance titration, mass spectrometry, Fourier transform infrared spectroscopy, Benesi-Hildebrand plot, and Job's plot in each case. The probe has some practical applications such as recovery of Al3+ from the drinking water sample, construction of the INHIBIT logic gate, and detection kits for Al3+ and TNP/DNP by simple paper test strips. The probe, H-PNAP, has successfully been applied to the detection of intracellular Al3+ and HF2 - ions in the human breast cancer cell, MDA-MB-468.

Trends in antibiotic resistance among major bacterial pathogens isolated from blood cultures tested at a large private laboratory network in India, 2008-2014.

OBJECTIVE: There have been no long-term studies on trends in antibiotic resistance (ABR) on a national scale in India. Using a private laboratory network, the ABR patterns of organisms most commonly associated with bacteremia, obtained from patients across India between 2008 and 2014, were examined. METHODS: A retrospective study of patient blood cultures collected over a 7-year period (January 1, 2008-December 31, 2014) was conducted. Data on the microorganism(s) identified and their antimicrobial susceptibility were obtained from SRL Diagnostics (Mumbai, India). RESULTS: Of 135268 blood cultures, 18695 (14%) had at least one identified pathogen. In addition to continual high rates of methicillin-resistant Staphylococcus aureus (MRSA; approximately 44.2%), high resistance to nalidixic acid among Salmonella Typhi (98%) was observed, and carbapenem resistance increased in both Escherichia coli (7.8% to 11.5%; p=0.332) and Klebsiella pneumoniae (41.5% to 56.6%; p<0.001). Carbapenem resistance was also stable and high for both Acinetobacter species (approximately 69.6%) and Pseudomonas aeruginosa (approximately 49%). Resistance was also detected to colistin in the Gram-negatives and to vancomycin and linezolid in S. aureus. CONCLUSION: Increasing resistance to antibiotics of last-resort, particularly among Gram-negatives, suggests an urgent need for new antibiotics and improved antimicrobial stewardship programs in India.

Pulmonary disease due to Mycobacterium massiliense.

We report a case of a patient suffering from multidrug-resistant pulmonary tuberculosis (MDR-PTB) who later developed an invasive infection of the respiratory tract with a rapidly growing non-tuberculous mycobacteria (NTM), recently identified as Mycobacterium massiliense, closely related to M. abscessus. To the best of our knowledge, this is the first case of M. massiliense infection being reported from India.