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The native extracellular matrix is continuously remodeled to form complex interconnected network structures that reversibly regulate stem cell behaviors. Both regulation and understanding of its intricate dynamicity can help to modulate numerous cell behaviors. However, neither of these has yet been achieved due to the lack of designing and modeling such complex structures with dynamic controllability. Here we report modularity-based mathematical modeling of extracellular matrix-emulating ligand inter-cluster connectivity using the graph theory. Increasing anisotropy of magnetic nano-blockers proportionately disconnects arginine-glycine-aspartic acid ligand-to-ligand interconnections and decreases the number of ligand inter-cluster edges. This phenomenon deactivates stem cells, which can be partly activated by linearizing the nano-blockers. Remote cyclic elevation of high-anisotropy nano-blockers flexibly generates nano-gaps under the nano-blockers and augments the number of ligand inter-cluster edges. Subsequently, integrin-presenting stem cell infiltration is stimulated, which reversibly intensifies focal adhesion and mechanotransduction-driven differentiation both in vitro and in vivo. Designing and systemically modeling extracellular matrix-mimetic geometries opens avenues for unraveling dynamic cell-material interactions for tissue regeneration., Competing Interests: Competing interests: Y.S.Z. consulted for Allevi by 3D Systems, sits on the scientific advisory board, and holds options of Xellar, neither of which, however, participated in or biased the work. All other authors declare no competing interests., (© 2024. The Author(s).)

The ability to gain spatiotemporal information, and in some cases achieve spatiotemporal control, in the context of drug delivery makes theranostic fluorescent probes an attractive and intensely investigated research topic. This interest is reflected in the steep rise in publications on the topic that have appeared over the past decade. Theranostic fluorescent probes, in their various incarnations, generally comprise a fluorophore linked to a masked drug, in which the drug is released as the result of certain stimuli, with both intrinsic and extrinsic stimuli being reported. This release is then signaled by the emergence of a fluorescent signal. Importantly, the use of appropriate fluorophores has enabled not only this emerging fluorescence as a spatiotemporal marker for drug delivery but also has provided modalities useful in photodynamic, photothermal, and sonodynamic therapeutic applications. In this review we highlight recent work on theranostic fluorescent probes with a particular focus on probes that are activated in tumor microenvironments. We also summarize efforts to develop probes for other applications, such as neurodegenerative diseases and antibacterials. This review celebrates the diversity of designs reported to date, from discrete small-molecule systems to nanomaterials. Our aim is to provide insights into the potential clinical impact of this still-emerging research direction.

Organic solvent nanofiltration (OSN) has evolved as a vital technological frontier with paramount significance in the separation and purification of organic solvents. Its implication is particularly prominent in industries such as pharmaceuticals, petrochemicals, and environmental remediation. This comprehensive review, meticulously navigates through the current state of research in OSN membranes, unveiling both the critical challenges and promising opportunities that beckon further exploration. The central focus of this review is on the unique utilization of covalent organic frameworks (COFs) and metal-organic frameworks (MOFs) in OSN membrane design, leveraging their distinctive structural attributes-tunable porosity, robust chemical stability, and molecular sieving capabilities. These qualities position them as exceptional candidates for crafting membranes tailored to the intricacies of organic solvent environments. Our investigation extends into the fundamental principles that render COFs and MOFs adept in OSN applications, dissecting their varied fabrication methods while offering insights into the advantages and limitations of each. Moreover, we address environmental and sustainability considerations in the use of COF and MOF-based OSN membranes. Furthermore, we meticulously present the latest advancements and innovations in this burgeoning field, charting a course toward potential future directions and emerging research areas. By underscoring the challenges awaiting exploration, this review not only provides a panoramic view of the current OSN landscape but also lays the groundwork for the evolution of efficient and sustainable OSN technologies, specifically harnessing the unique attributes of COFs and MOFs.

Recently, newly developed carbon-based nanomaterials known as carbon dots (CDs) have generated significant interest in nanomedicine. However, current knowledge regarding CD research in the biomedical field is still lacking. An overview of the most recent development of CDs in biomedical research is given in this review article. Several crucial CD applications, such as biosensing, bioimaging, cancer therapy, and antibacterial applications, are highlighted. Finally, CD-based biomedicine's challenges and future potential are also highlighted to enrich biomedical researchers' knowledge about the potential of CDs and the need for overcoming various technical obstacles.

Nanomedicines for drug delivery and imaging-guided cancer therapy is a rapidly growing research area. The unique properties of nanomedicines have a massive potential in solving longstanding challenges of existing cancer drugs, such as poor localization at the tumor site, high drug doses and toxicity, recurrence, and poor immune response. However, inadequate biocompatibility restricts their potential in clinical translation. Therefore, advanced nanomaterials with high biocompatibility and enhanced therapeutic efficiency are highly desired to fast-track the clinical translation of nanomedicines. Intrinsic properties of nanoscale covalent organic frameworks (nCOFs), such as suitable size, modular pore geometry and porosity, and straightforward post-synthetic modification via simple organic transformations, make them incredibly attractive for future nanomedicines. The ability of COFs to disintegrate in a slightly acidic tumor microenvironment also gives them a competitive advantage in targeted delivery. This review summarizes recently published applications of COFs in drug delivery, photo-immuno therapy, sonodynamic therapy, photothermal therapy, chemotherapy, pyroptosis, and combination therapy. Herein we mainly focused on modifications of COFs to enhance their biocompatibility, efficacy and potential clinical translation. This review will provide the fundamental knowledge in designing biocompatible nCOFs-based nanomedicines and will help in the rapid development of cancer drug carriers and theranostics., (© 2022 The Authors.)

Elucidating the underlying photochemical mechanisms of action (MoA) of photodynamic therapy (PDT) may allow its efficacy to be improved and could set the stage for the development of new classes of PDT photosensitizers. Here, we provide evidence that "photoredox catalysis in cells," wherein key electron transport pathways are disrupted, could constitute a general MoA associated with PDT. Taking the cellular electron donor nicotinamide adenine dinucleotide as an example, we have found that well-known photosensitizers, such as Rose Bengal, BODIPY, phenoselenazinium, phthalocyanine, and porphyrin derivatives, are able to catalyze its conversion to NAD + . This MoA stands in contrast to conventional type I and type II photoactivation mechanisms involving electron and energy transfer, respectively. A newly designed molecular targeting photocatalyst (termed CatER) was designed to test the utility of this mechanism-based approach to photosensitizer development. Photoexcitation of CatER induces cell pyroptosis via the caspase 3/GSDME pathway. Specific epidermal growth factor receptor positive cancer cell recognition, high signal-to-background ratio tumor imaging (SBRTI = 12.2), and good tumor growth inhibition (TGI = 77.1%) are all hallmarks of CatER. CatER thus constitutes an effective near-infrared pyroptotic cell death photo-inducer. We believe the present results will provide the foundation for the synthesis of yet-improved phototherapeutic agents that incorporate photocatalytic chemistry into their molecular design.

Porous organic polymers (POPs) are prepared by crosslinked polymerization of multidimensional rigid aromatic building blocks. Generally, POPs can be classified into crystalline covalent organic frameworks (COFs) and other poorly crystalline or amorphous porous polymers. Due to their remarkable intrinsic properties, such as high porosity, stability, tunability, and presence of numerous building blocks, several new POPs are being developed for application across various scientific fields. The essential sensitive functional groups needed for specific applications are not sustained under harsh POP preparation conditions. The recently developed post-synthetic modification (PSM) strategies for POPs have enabled their advanced applications that are otherwise restricted. Owing to the advanced PSM strategies POPs have experienced a blossoming resurgence with diverse functions, particularly in biomedical applications, such as bioimaging tools, drugs, enzymes, gene or protein delivery systems, phototherapy, and cancer therapy. This tutorial review focuses on the recently developed PSM strategies for POPs, especially for biomedical applications, and their future perspectives as promising bioapplicable materials.

Finding a personalized nano theranostics solution, a nanomedicine for cancer diagnosis and therapy, is among the top challenges of current medicinal science. Porous organic polymers (POPs) are permanent porous organic materials prepared by linking relatively rigid multidimensional organic building blocks. POP nanoparticles have a remarkable advantage for cancer theranostics owing to their specific physicochemical characteristics such as high surface area, convincing pore size engineering, stimuli-responsive degradability, negligible toxicity, open covalent post-synthesis modification possibilities etc. POPs have crystalline and non-crystalline characteristics; crystalline POPs are popularly known as covalent organic frameworks (COFs), and have shown potential application across research areas in science. The early research and development on theranostics applications of nanoscale POPs has shown tremendous future potential for clinical translation. This tutorial review highlights the recently developed promising applications of nPOPs in drug loading, targeted delivery, endogenous and exogenous stimuli-responsive release, cancer imaging and combination therapy, regardless of their crystalline and poorly crystalline properties. The review will provide a platform for the future development and clinical translation of nPOPs by solving fundamental challenges of cancer nanomedicines in drug loading efficiency, size-optimization, biocompatibility, dispersibility and cell uptake ability.

Solar light-driven fuel production from carbon dioxide using organic photocatalysts is a promising technique for sustainable energy sources. Band gap engineering in sustainable organic photocatalysts for improving efficiency and fulfilling the requirements is highly anticipated. Here, we present a new strategy to engineer the band gap in covalent organic framework (COF) photocatalysts by varying the push-pull electronic effect. To implement this strategy, we have designed and synthesized four different COFs using a tripodal amine 4,4',4″-(1,3,5-triazine-2,4,6-triyl)tris(([1,1'-biphenyl]-4-amine)) [Ttba] with 1,3,5-triformylbenzene ( COF-1 ), 2,4,6-triformylphloroglucinol ( COF-2 ), 2,4,6-triformylphenol ( COF-3 ), and 2,4,6-triformylresorcinol ( COF-4 ). On varying the number of hydroxyl units in the aldehyde precursor, the resulting COFs allow the fine-tuning of their band gap and band edge positions and result in different morphologies with varying surface areas. The enhanced optical properties of COF-3 and COF-4 with very suitable band gaps of 2.02 and 1.95 eV, respectively, enable them to demonstrate a high-efficiency photobiocatalytic system for NADH photoregeneration and enhanced visible light-driven formic acid production at a rate of 226.3 μmol g -1 in 90 min. The triazine core enables efficient charge separation, while the hydroxyl groups induce an electronic push-pull effect, regulating their photocatalytic efficiency. The results demonstrated the morphology-guided enhanced surface area and dual keto-enol tautomerism-induced push-pull effect in asymmetrical charge distribution as key features in the fine-tuning of the photocatalysts.

Ru(II)-metallomacrocycles containing 4-pyridyl-1,2,3-triazole moiety were realized by coordination-driven self-assembly. All new compounds were characterized by electrospray ionisation mass spectrometry, elemental analysis, and 1 H and 13 C NMR spectroscopic techniques. The molecular structure of metallomacrocycle 8 was determined by single-crystal X-ray crystallography. The anticancer activities of metallomacrocycles 5 - 8 were evaluated by cytotoxicity, cell cycle analysis, and related protein expression. Metallomacrocycle 7 showed the highest cytotoxicity in HepG2 human hepatocellular carcinoma cells. In addition, apoptotic HepG2 cells were analyzed when metallomacrocycle 7 was treated. Our results suggest that metallomacrocycle 7 induces liver cancer cell death by increasing apoptosis and cell cycle arrest and that it has potential use as an agent for the treatment of human hepatocellular carcinoma., Competing Interests: The authors declare no competing financial interest.

Here we report the synthesis of a linear [3]catenane comprised of three interlocking rings, by coordination-driven self-assembly. Naphthalene-based acceptor A1 and triazole-based donor L1 were utilized for the self-assembly reaction, which facilitated the formation of linear [3]catenane topology through synergistic non-covalent intercycler interactions (π-π, CH-π and CH-N).

We report herein on the design, synthesis and biological activity of Ru-based self-assembled supramolecular bowls as a potent anticancer therapeutic in human hepatocellular cancer. The potent complex induces production of reactive oxygen species (ROS) by higher fatty acid β-oxidation and down-regulation of glucose transporter-mediated pyruvate dehydrogenase kinase 1 via reduced hypoxia-inducible factor 1α. Also, overexpressed acetyl-CoA activates the tricarboxylic acid cycle and the electron transport system and induces hypergeneration of ROS. Finally, ROS overexpressed through this pathway leads to apoptosis. Furthermore, we demonstrate that the naphthalene derived molecular bowl activates classical apoptosis via crosstalk between the extrinsic and intrinsic signal pathway. Our work into the mechanism of Ru-based self-assembled supramolecular bowls can provide valuable insight into the potential for use as a promising anticancer agent.

Lipopolysaccharide (LPS) is a major virulence factor of Gram-negative bacteria playing a major role in stimulating protective immune response in mammalian host. However, in many gram-negative bacterial infections, LPS also elicits immunopathology by inducing excessive inflammatory changes. P. multocida (Pm), a gram-negative bacterium, causes acute lung inflammation and fatal septicemic disease in animals. However, the effects of Pm LPS on host cells are little known. In this study, LPS isolated from three different serotypes (B:2, A:1 and A:3) of Pm were individually tested in vitro to assess the response of bovine leukocytes. Pm LPS induced cell proliferation and cell death of leukocytes, in a dose- and time-dependent manner. In these cells, mitochondrial dysfunction and caspase activation mediate cell death., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Molecular knots have become highly attractive to chemists because of their prospective properties in mimicking biomolecules and machines. Only a few examples of molecular knots from the billions tabulated by mathematicians have been realized and molecular knots with more than eight crossings have not been reported to date. We report here the coordination-driven [8+8] self-assembly of a higher-generation molecular knot comprising as many as sixteen crossings. Its solid-state X-ray crystal structure and multinuclear 2D NMR findings confirmed its architecture and topology. The formation of this molecular knot appears to depend on the functionalities and geometries of donor and acceptor in terms of generating appropriate angles and strong π-π interactions supported by hydrophobic effects. This study shows coordination-driven self-assembly offers a powerful potential means of synthesizing more and more complicated molecular knots and of understanding differences between the properties of knotted and unknotted structures., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Three-dimensional molecular architectures self-assembled with tripodal and tetratopic donors are valuable because of their encapsulation properties. Here, we present Co(I)-Fe(II)-Pd(II) heterotrimetallic trifacial barrel 1, which was self-assembled using a newly synthesized tetratopic donor [CpCo(CbR 4 )] [L; Cp = cyclopentadienyl, Cb = cyclobudiene, and R = 4-(4-pyridylphenyl)] and a 90° acceptor [ cis-(dppf)Pd(OTf) 2 ] (A1; dppf = (diphenylphosphino)ferrocene and OTf = CF 3 SO 3 - ). The heterotrimetallic barrel 1 exhibited selective 1:1 interaction with a N, N'-dimethyl-1,4,5,8-naphthalenetetracarboxylic diimide guest, as revealed by 1 H NMR analysis. The self-assembly of donor L with two other Ru(II)-based 180° acceptors [( p-cymene) 2 Ru 2 (OO∩OO)(OTf) 2 ] [OO∩OO = 6,11-dioxido-5,12-naphthacenedione (A2) and oxalate (A3)] resulted in tetragonal-prismatic cages. Self-assembly using the longer acceptor A2 provided rare isomers of a tetragonal-prismatic cage by varying the orientation of the cyclopentadienyl moiety out-out (2 a ) or out-in (2 b ) of the cavity, whereas self-assembly using the shorter acceptor A3 selectively resulted in the tetragonal-prismatic cage 3. The three-dimensional molecular architectures 1-3 were characterized by combined spectroscopic and elemental analyses. The structures of molecular barrel 1 and prismatic cage 3 were elucidated by single-crystal X-ray analysis.

Coordination-driven self-assembly of m-bis[3-(4-pyridyl)pyrazolyl]xylene (L) and [(p-cymene) 2 Ru 2 (OO∩OO) 2 (OTf) 2 ] (A 1 ) (OO∩OO = 6,11-dioxido-5,12-naphthacenedione) in methanol resulted in a mixture of [2]catenane 1 and macrocycle 2, and self-assembly in nitromethane resulted in pure macrocycle 2, whereas the coordination-driven self-assembly of L and similar acceptors [(p-cymene) 2 Ru 2 (OO∩OO) 2 (OTf) 2 ] [OO∩OO = 5,8-dioxido-1,4-naphthoquinonnato (A 2 ); 2,5-dioxido-1,4-benzoquinonato (A 3 ); oxalato (A 4 )] resulted in the formations of monomeric macrocycles 3-5, respectively. All self-assembled macrocycles were obtained in excellent yields (>90%) as triflate salts and were fully characterized by multinuclear NMR, elemental analysis, and electrospray ionization mass spectrometry (ESI-MS). The structures of [2]catenane 1 and macrocycles 5 were confirmed by single-crystal X-ray diffraction analysis. The X-ray structure of 1 confirmed an edge-to-face interaction between the tetracene moiety in parallel-displaced π-π stacks (3.5 Å), and CH···π (2.5 Å) stabilizes the [2]catenane topology. Macrocycles 2-5 were assessed for anticancer activities using human cancer cell lines of different origins, and the macrocycle 3 was found to exhibit the best inhibitory effect and to do so in a dose-dependent manner. Further examination with the Tali apoptosis assay suggested the growth inhibitory effect of 3 involved the induction of the programmed cell death, and this suggestion was supported by observations of PARP and caspase 3 cleavage after treating cells with 3. In addition, exposure to 3 increased the expression of Bax and repressed the expression of Bcl-2, thus indicating the involvement of macrocycle 3 upstream of Bax and Bcl-2 in the apoptotic signaling pathway. Macrocycle 3 also tended to repress metastasis as evidenced by changes in the transcriptional expressions E- and N-cadherin (markers of metastasis). Furthermore, a stability assay demonstrated macrocycle 3 remained stable at high concentration.

This paper reports the formation of unprecedented iridium(iii)-derived topological macrocycles. Discrete molecular Borromean rings 1 and 3 in pure form are synthesized via coordination-driven self-assembly of an acceptor [(Cp*Ir) 2 (OO∩OO)](OTf) 2 (Cp* = pentamethylcyclopentadienyl, OO∩OO = 6,11-dioxido-5,12-naphthacenedione) (A) with dipyridyl donors 1,4-bis(4-pyridinylethynyl)benzene (L 1 ) and 2,5-bis(4-pyridinylethynyl)thiophene (L 2 ) respectively in methanol. Self-assembly using the same acceptor under similar conditions with two other donors 9,10-bis(4-pyridinylethynyl)anthracene (L 3 ) and 1,4-di(4-pyridinylethynyl)buta-1,3-diyne (L 4 ) resulted in [2]catenane 5 and non-catenane ring-in-ring topological macrocycle 7 respectively. Rectangular macrocycles 2, 4, 6, and 8 were respectively obtained when the self-assembly of acceptor A with one of the donors L 1 -L 4 was carried out under dilute conditions in nitromethane or methanol. All these new macrocycles were characterized by 1 H and 13 C NMR, 2D NMR, ESI-MS and elemental analyses. Single crystal X-ray structures of Borromean rings 1 and 3, and ring-in-ring macrocycle 7 revealed that the length and functionality of donors enabling CHπ and ππ interactions govern the topology.

Three new cobalt-ruthenium heterometallic molecular rectangles, 1-3, were synthesized through the coordination-driven self-assembly of a new cobalt sandwich donor, (η 5 -Cp)Co[C 4 -trans-Ph 2 (4-Py) 2 ] (L; Cp: cyclopentyl; Py: pyridine), and one of three dinuclear precursors, [(p-cymene) 2 Ru 2 (OO∩OO) 2 Cl 2 ] [OO∩OO: oxalato (A 1 ), 5,8-dioxido-1,4-naphthoquinone (A 2 ), or 6,11-dioxido-5,12-naphthacenedione (A 3 )]. All of the self-assembled architectures were isolated in very good yield (92-94 %) and were fully characterized by spectroscopic analysis; the molecular structures of 2 and 3 were determined by single-crystal X-ray diffraction analysis. The anticancer activities of bimetallic rectangles 1-3 were evaluated with a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, an autophagy assay, and Western blotting. Rectangles 1-3 showed higher cytotoxicity than doxorubicin in AGS human gastric carcinoma cells. In addition, the autophagic activities and apoptotic cell death ratios were increased in AGS cells by treatment with 1-3; the rectangles induced autophagosome formation by promoting LC3-I to LC3-II conversion and apoptotic cell death by increasing caspase-3/7 activity. Our results suggest that rectangles 1-3 induce gastric cancer cell death by modulating autophagy and apoptosis and that they have potential use as agents for the treatment of human gastric cancer., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Molecular Borromean rings (BRs) is one of the rare topology among interlocked molecules. Template-free synthesis of BRs via coordination-driven self-assembly of tetracene-based Ru(II) acceptor and ditopic pyridyl donors is reported. NMR and single-crystal XRD analysis observed sequential transformation of a fully characterized monomeric rectangle to molecular BRs and vice versa. Crystal structure of BRs revealed that the particular topology was enforced by the appropriate geometry of the metallacycle and multiple parallel-displaced π-π interactions between the donor and tetracene moiety of the acceptor. Computational studies based on density functional theory also supported the formation of BRs through dispersive intermolecular interactions in solution.

A molecular Solomon link was synthesized in high yield through the template-free, coordination-driven self-assembly of a carbazole-functionalized donor and a tetracene-based dinuclear ruthenium(II) acceptor. The doubly interlocked topology was realized by a strategically chosen ligand which was capable of participating in multiple CH⋅⋅⋅π and π-π interactions, as evidenced from single-crystal X-ray analysis and computational studies. This method is the first example of a two-component self-assembly of a molecular Solomon link using a directional bonding approach. The donor alone was not responsible for the construction of the Solomon link, and was confirmed by its noncatenane self-assemblies obtained with other similar ruthenium(II) acceptors., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Nano-sized multinuclear ruthenium complexes have rapidly emerged as promising therapeutic candidates with unique anticancer activities. Here, we describe the coordination-driven self-assembly and anticancer activities of a set of three organometallic tetranuclear Ru(II) molecular bowls. [2+2] Coordination-driven self-assembly of 3, 6-bis(pyridin-3- ylethynyl) phenanthrene (bpep) (1) and one of the three dinuclear arene ruthenium clips, [(η6-p-iPrC6H4Me)2Ru2-(OO\OO)][OTf]2 (OO\OO =2, 5-dioxido-1, 4-benzoquinonato, OTf = triflate) (2), 5, 8-dioxido-1, 4-naphthoquinonato (3), or 6, 11-dioxido-5, 12-naphthacenediona (4), resulted in three molecular bowls 5-7 of general formula [{(η6-p-iPrC6H4Me)2Ru2-(OO\OO)}2(bpep)2][OTf]4. All molecular bowls were obtained as triflate salts in very good yields (>90%) and were fully characterized using multinuclear nuclear magnetic resonance (NMR), electrospray ionization-mass spectrometry (ESI-MS), and elemental analysis. The structure of the representative molecular bowl 5 was confirmed by single-crystal X-ray diffraction analysis. The anticancer activities of molecular bowls 5-7 were determined by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide, autophagy, and Western blot analysis. Bowl 6 showed the strongest cytotoxicity in AGS human gastric carcinoma cells and was more cytotoxic than doxorubicin. In addition, autophagic activity and the ratio of apoptotic cell death increased in AGS cells by treatment with bowl 6. Bowl 6 also induced autophagosome formation via upregulation of p62 and promotion of the conversion of LC3-I to LC3-II. Moreover, bowl 6 promoted apoptotic cell death through downregulation of Akt/mTOR activation, followed by increased caspase-3 activity. These results suggest that bowl 6 induces gastric cancer cell death via modulation of autophagy and apoptosis. Bowl 6 is a potent anticancer agent and a potential treatment for human gastric cancer that merits further study.

The coordination-driven self-assembly of an anthracene-functionalized ditopic pyridyl donor and a tetracene-based dinuclear Ru(II) acceptor resulted in an interlocked metalla[2]catenane, [M2L2]2, in methanol and a corresponding monorectangle, [M2L2], in nitromethane. Subsequently, guest template, solvent, and concentration effects allowed the self-assembly to be reversibly fine-tuned among monorectangle and catenane structures.

A hollow iridium-cornered prismatic cage was self-assembled without the assistance of any template. The cage was found to be capable of encapsulating heteroguest's triplet in its perfect sized cavity, producing the first demonstration of quintuple structure by an octahedral metal cornered prismatic cage.

The reaction of (β-phenylethynyl)pentafluorocyclotriphosphazene, [(PhC≡C)(F)PN](PNF2)2, with Fe2(CO)9 in refluxing hexane resulted in five new compounds, namely, [Fe(CO)2{η(2):η(2)-2,4-(P3N3F5)2Ph2C4Fe(CO)3}-μ-CO] (1), [Fe(CO)2{η(2):η(2)-2,5-(P3N3F5)2Ph2C4Fe(CO)3}-μ-CO] (2), [Fe(CO)2{η(5)-2,5-(P3N3F5)2Ph2C4CO}C(Ph)═C(P3N3F5)](3), [Fe(CO)3{η(2):η(2)-2,4-(P3N3F5)2Ph2C4CO] (4), and [Fe(CO)3{η(2):η(2)-2,5-(P3N3F5)2Ph2C4CO] (5). While compounds 1, 2, 4, and 5 have five-membered ferracyclopentadiene or cyclopentadienone rings coordinated to the Fe(CO)3 unit in the η(2):η(2) mode, compound 3 has a 2,5-cyclopentadienone ring attached to an Fe(CO)2(P3N3F5)C═C(Ph) unit, where Fe is η(5)-bonded to the cyclopentadienone ring, and the carbon that is α to the phenyl unit of the Fe(CO)2(P3N3F5)C═C(Ph) group is σ-bonded to the oxygen atom of the cyclopentadienone ring. Formation of five-membered cyclic compounds having two fluorophosphazene units on the vicinal carbon atoms of C4R2R'2Y rings was not observed in this reaction. No examples of Fe(CO)3-bound cyclobutadiene complexes were also isolated from this reaction. A similar reaction in the presence of trimethylamine N-oxide, NMe3O, was found to proceed at -20 °C with the formation of compounds 4 and 5 only. In contrast to the Fe2(CO)9 reaction, a reaction of alkyne-derived pentafluorocyclotriphosphazenes, [(RC≡C)(F)PN](PNF2)2 [R = Ph, Fe(C5H5)2] with the molybdenum complex Cp(CO)3Mo-Mo(CO)3Cp (Cp = cyclopentadienyl) in refluxing toluene resulted in the simple tetrahedral clusters Cp(CO)2Mo(P3N3F5)C-C(Ph)Mo(CO)2Cp (6) and Cp(CO)2Mo(P3N3F5)C-C(Fc)Mo(CO)2Cp (7) (Fc = ferrocenyl). A similar reaction of Cp(CO)3Mo-Mo(CO)3Cp with butadiyne-derived fluorophosphazenes, [(RC≡C-C≡C)(F)PN](PNF2)2 [R = Ph, Fe(C5H5)2], yielded the tetrahedral clusters Cp(CO)2Mo(P3N3F5)C-C(C≡CPh)Mo(CO)2Cp (8) and Cp(CO)2Mo(P3N3F5)C-C(C≡CFc)Mo(CO)2Cp (9) with the tetrahedral Mo2C2 unit forming exclusively with the alkyne unit of the butadiyne group bound to the cyclophosphazene ring. The crystal structures and infrared spectral data of these molybdenum clusters showed the presence of a semibridging carbonyl on one of the molybdenum units. All new compounds were characterized by IR, NMR [(1)H, (13)C{(1)H}, (31)P{(1)H}, and (19)F{(1)H}] and high-resolution mass spectrometry studies. Compounds 2, 3, 5, and 7-9 were also structurally characterized using single-crystal X-ray diffraction studies.

The interaction between Mycobacterium avium subsp. paratuberculosis (Map) and macrophages is a complex process to maximize the chances of their respective survival. Previous studies have shown that Map induces cell death in macrophages, but the mechanism is not known. In the present study, we investigated the mechanism by which Map induces cell death in bovine macrophages using the fluorescent and electron microscopic techniques. The macrophages infected with an equal number of Map (i.e., multiplicity of infection, MOI=1) showed no changes of cell death, but those macrophages infected at MOI=10 showed the morphological changes consistent with apoptosis. Strikingly, the macrophages infected by Map at MOI=50 showed the changes of apoptosis and necrosis. The Map-induced apoptosis was a caspase-dependent mechanism at MOI=10 while it was caspase- and nitric oxide-independent at MOI=50. The results of the present study suggest that the mitochondrial damage following Map infection initiates the cell death processes in macrophages., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A novel protecting group directed diversity leading to the synthesis of bridged bicyclic and six-membered iminocyclitols from a common carbohydrate derived diamino triol under Mitsunobu conditions is reported. When the intramolecular cyclization of benzoyl derivative 16 was carried out under Mitsunobu conditions, an unprecedented one-pot domino intramolecular "cyclization-N→O benzoyl migration-cyclization" reaction sequence occurred resulting in the formation of a chiral 2,6-diazabicyclo[3.2.1]octane-4,8-diol 21 in high yield. The structure of this novel bridged bicyclic compound was established through detailed NMR studies and single crystal X-ray analysis. On the other hand, the tert-butyldimethylsilyl derivative of the same substrate afforded protected 6-amino-1,6-dideoxy-l-gulonojirimycin 32 as the sole product under identical conditions. An attempt has been made to explain this difference in their reactivity through conformational analysis. The glycosidase inhibition studies of new compounds reported in this manuscript revealed that these molecules display moderate but selective inhibition against β-N-acetylhexosaminidase.

The reaction of 1,4-diphenylbutadiyne along with diphenylacetylene, when carried out with η(5)-[MeOC(O)Cp]Co(PPh(3))(2) resulted in the cyclobutadiene linked dimeric cobalt sandwich compound {[η(5)-MeOC(O)Cp]Co(η(4)-C(4)Ph(3))}(2) (1) along with the known monomeric compound η(5)-[MeOC(O)Cp]Co(η(4)-C(4)Ph(4)). Compound 1, on treatment with KOH gave the dicarboxylic acid {η(5)-[HOC(O)Cp]Co(η(4)-C(4)Ph(3))}(2) (2) which on reaction with oxalyl chloride followed by (S)-2-amino-3-methyl-1-butanol, triethylamine and mesylchloride was converted to the bis(cobalt oxazoline) based chiral ligand {[η(5)-(4-iPr-2-Ox)Cp]Co(η(4)-C(4)Ph(3))}(2) (3) (Ox = Oxazolinyl). Compound 3 on reaction with palladium acetate gave an NCN bound bis(cobalt-oxazolinyl) palladacycle {[η(5)-(4-iPr-2-Ox)Cp]Co(η(4)-C(4)Ph(3))}(2)PdOAc (4) having both the oxazolinyl groups bound to the palladium in a chelate mode and one of the Cp rings forming a five membered [C,N] palladacycle. Reaction of 4 with aq. NaCl resulted in the chloro analogue of 4, {[η(5)-(4-iPr-2-Ox)Cp]Co(η(4)-C(4)Ph(3))}(2)PdCl (5). A nonchiral bidentate bis cobalt oxazoline ligand {[η(5)-(Ox)Cp]Co(η(4)-C(4)Ph(3))}(2) (6) was also prepared by replacing (S)-2-amino-3-methyl-1-butanol with 2-amino-1-ethanol in the procedure used for the preparation of 3. A reaction of 1,4-diphenylbutadiyne, diphenylacetylene and (η(5)-Cp)Co(PPh(3))(2) resulted in the dimer [(η(5)-Cp)Co(η(4)-C(4)Ph(3))](2) (7) and small amounts of a trimer [(η(5)-Cp)Co(η(4)-C(4)Ph(3))](2)[(η(5)-Cp)Co(η(4)-C(4)Ph(2))] (8) having no substituents on the Cp rings. All the compounds except 2 were structurally characterized. Structural studies on palladacycles 4 and 5 showed interesting anagostic C-H···Pd interactions between the ortho hydrogen of one of the Cp rings and the palladium centre. Preliminary studies carried out on the palladacycles showed promising catalytic activity in the asymmetric rearrangement of trichloroacetimidates.

The first examples of ring-closing metathesis (RCM) reactions of a series of terminal alkene-derived cyclic phosphazenes have been carried out. The tetrakis-, hexakis-, and octakis(allyloxy)cyclophosphazenes (NPPh(2))(NP(OCH(2)CH=CH(2))(2))(2) (1), N(3)P(3)(OCH(2)CH=CH(2))(6) (2), and N(4)P(4)(OCH(2)CH=CH(2))(8) (3) and the tetrakis(allyloxy)-S-phenylthionylphosphazene (NS(O)Ph)[NP(OCH(2)CH=CH(2))(2)](2) (4) were prepared by the reactions of CH(2)=CHCH(2)ONa with the cyclophosphazenes (NPPh(2))(NPCl(2))(2), N(3)P(3)Cl(6), and N(4)P(4)Cl(8) and the S-phenylthionylphosphazene (NS(O)Ph)(NPCl(2))(2). The reactions of 1-4 with Grubbs first-generation olefin metathesis catalyst Cl(2)Ru=CHPh(PCy(3))(2) resulted in the selective formation of seven-membered di-, tri-, and tetraspirocyclic phosphazene compounds (NPPh(2))[NP(OCH(2)CH=CHCH(2)O)](2) (5), N(3)P(3)(OCH(2)CH=CHCH(2)O)(3) (6), and N(4)P(4)(OCH(2)CH=CHCH(2)O)(4) (7) and the dispirocyclic S-phenylthionylphosphazene compound (NS(O)Ph)[NP(OCH(2)CH=CHCH(2)O)](2) (8). X-ray structural studies of 5-8 indicated that the double bond of the spiro-substituted cycloalkene units is in the cis orientation in these compounds. In contrast to the reactions of 1-4, RCM reactions of the homoallyloxy-derived cyclophosphazene and thionylphosphazene (NPPh(2))[NP(OCH(2)CH(2)CH=CH(2))(2)](2) (9) and (NS(O)Ph)[NP(OCH(2)CH(2)CH=CH(2))(2)](2) (10) with the same catalyst resulted in the formation of 11-membered diansa compounds NPPh(2)[NP(OCH(2)CH(2)CH=CHCH(2)CH(2)O)](2) (11) and (NS(O)Ph)[NP(OCH(2)CH(2)CH=CHCH(2)CH(2)O)](2) (13) and the intermolecular doubly bridged ansa-dibino-ansa compounds 12 and 14. The X-ray structural studies of compounds 11 and 13 indicated that the double bonds of the ansa-substituted cycloalkene units are in the trans orientation in these compounds. The geminal bis(homoallyloxy)tetraphenylcyclotriphosphazene [NPPh(2)](2)[NP(OCH(2)CH(2)CH=CH(2))(2)] (15) upon RCM with Grubbs first- and second-generation catalysts gave the spirocyclic product [NPPh(2)](2)[NP(OCH(2)CH(2)CH=CHCH(2)CH(2)O)] (16) along with the geminal dibino-substituted dimeric compound [NPPh(2)](2)[NP(OCH(2)CH(2)CH=CHCH(2)CH(2)O)(2)PN][NPPh(2)](2) (17) as the major product. The dibino compound 17, upon reaction with the Grubbs second-generation catalyst, was found to undergo a unique ring-opening metathesis reaction, opening up the bino bridges and partially converting to the spirocyclic compound 16.

Mice were experimentally infected with Pasteurella multocida serotype A1 to study the cytokine profiles, host cell apoptosis and sequential pathology at different hours of post-infection. Infected mice were dull, anorectic and depressed. A transient leukocytopenia followed by progressive leukocytosis was observed in the course of infection. Serum cytokine profiles showed significantly (P<0.01) higher amount of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and mouse KC) in the infected mice when compared to control mice. The circulating lymphocytes were apoptotic on annexin V staining. Apoptotic nuclei were detected in splenocytes, hepatocytes and infiltrating leukocytes of the lungs on TUNEL staining. The lungs were grossly congested and hemorrhagic, and showed infiltration with polymorphonuclear cells at early and mononuclear cells in the late hours of infection. Alveolar epithelia, inter-alveolar septa and capillary endothelium of the lungs showed ultrastructural changes. Liver had degenerative changes in histological and ultrathin sections., (Published by Elsevier India Pvt Ltd.)

Some bicyclic C-fused chlorinated tetrahydrofuro[3,2-c]azetidin-2-ones were prepared by a fairly general route involving Staudinger reaction of allylic/propargylic imidates with dichloroketene followed by highly diastereoselective CuCl/PMDETA-catalyzed 5-exo-trig/dig chlorine atom transfer radical cyclization. An oxepan-fused β-lactam was also prepared similarly by 7-endo-trig cyclization. Synthetic application of the side chain chlorine atom of the products was demonstrated by its substitution in one of the products with azide followed by azide-alkyne click reaction with phenylacetylene to obtain a 1,2,3-triazolyltetrahydrofuro[3,2-c]azetidin-2-one.

Foot-and-mouth disease (FMD), the most contagious animal disease, is associated with persistent viral infection in ruminants, despite the induction of systemic immune response. The present study was performed to decipher the relation between the persistent FMD virus (FMDV) infection and cellular immune response in Indian cattle (Bosindicus) following experimental inoculation of FMDV Asia 1. Persistent viral infection (carriers) was detected by antigen capture RT-PCR on the oesophageal-pharyngeal fluid. Viral excretion was found to be intermittent and strongly variable among the persistently infected Indian cattle. Lymphocyte proliferative (LP) response, assessed as reactivity of peripheral blood mononuclear cells to FMDV Asia 1 antigen (Ag) was of low magnitude indicating a weak primary cellular immune response following infection. LP response to FMDV Ag was higher among the non-carriers than carriers of FMDV Asia 1. An enhanced LP response was associated with the lack of virus shedding in the OPF. The findings of this study are suggestive of relationship between cellular immune response and virus excretion during persistence of FMDV Asia 1 in infected cattle., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The reactions of lithiated ethynylferrocene, FcC[triple bond]CLi (Fc = ferrocenyl), with fluoro- and chlorocyclotriphosphazenes, N(3)P(3)X(6) (X = F, Cl), resulted in the formation of stable mono-FcC[triple bond]CP(3)N(3)X(5) [X = F (1), Cl (2)] and geminal bis[(FcC[triple bond]C)(2)PN](X(2)PN)(2) [X = F (3), Cl (4)], ethynylferrocene-substituted cyclophosphazenes. The reactions of 1 and 2 with CpCo(COD) were found to differ in the nature of the sandwich compounds (eta(5)-Cp)Co{eta(4)-C(4)[Fc(2)(N(3)P(3)X(5))(2)]} formed. While the fluorophosphazene-derived compound 1 yielded both cis and trans isomers of the cyclobutadiene complexes 5 and 6, the chlorophosphazene-derived compound 2 was found to give only the trans-cyclophosphazene-disubstituted cyclobutadiene compound 7. The reaction of 3 with CpCo(COD) was found to give the compound (eta(5)-Cp)Co{eta(4)-C(4)-1,3-(Fc)(2)-2,4-[NP(C[triple bond]CFc)(NPF(2))(2)](2)} (8) having one of the alkyne units of the cyclophosphazene moiety of 3 remaining unreacted even after reaction with an excess of CpCo(COD). The first click reactions of ethynylferrocene-derived cyclophosphazenes have been carried out by reacting 1 and 4 with benzyl azide, resulting in novel mono- and disubstituted cyclophosphazene-derived 1,2,3-triazoles. The reaction of 1 with benzyl azide yielded two positional isomers of the 1,2,3-triazole, (1-PhCH(2), 4-Fc, 5-P(3)N(3)F(5))C(2)N(3) (9), and (1-PhCH(2), 4-P(3)N(3)F(5), 5-Fc)C(2)N(3) (10), with the latter having a benzyl group in the vicinity of the ferrocene unit as the major product. A similar reaction of 4 with benzyl azide was found to yield five triazole-based products, with two, [(1-PhCH(2)-4-FcC(2)N(3))(C[triple bond]CFc)PN](PNCl(2))(2) (11) and [(1-PhCH(2)-5-Fc-C(2)N(3))(C[triple bond]CFc)PN](PNCl(2))(2) (12), having one unreacted alkyne unit in the molecule. Among the bis-triazole-derived chlorophosphazenes [(1-PhCH(2)-4-Fc-C(2)N(3))(2)PN](PNCl(2))(2) (13), [(1-PhCH(2)-4-Fc-C(2)N(3))(1-PhCH(2)-5-Fc-C(2)N(3))PN](PNCl(2))(2)] (14), and [(1-PhCH(2)-5-Fc-C(2)N(3))(2)PN](PNCl(2))(2) (15), the unsymmetrically disubstituted bis-triazole compound 14 was found to be the major product. The fluorophosphazene-derived triazole 10 was found to readily form a disubstituted square-planar complex trans-[(1-PhCH(2)-4-P(3)N(3)F(5)-5-Fc)C(2)N(3)](2)PdCl(2) (16) with PdCl(2)(PhCN)(2). All of the new ferrocene- and cyclophosphazene-derived compounds except 8 and 12 have also been structurally characterized.

Despite their potential role in the spread of foot-and-mouth disease (FMD), the immune response and viral persistence in FMD virus (FMDV)-infected Indian buffaloes (Bubalus bubalis) have been unexplored. We found similar kinetics of neutralizing antibody responses in the sera and secretory fluids of buffaloes following experimental FMDV Asia 1 infection, but the lymphocyte-proliferative response in infected buffaloes was of low magnitude. Despite inducing a significant systemic and secretory immune response, viral persistence seems to be a common outcome in buffaloes following FMDV Asia 1 infection, which is associated with a weak cellular immune response.

Humoral and mucosal (secretory antibody)immune response to FMDV type Asia 1 in cattle was analyzed after vaccination and infection using virus neutralizing test (VNT). Vaccination (1/16th the usual dose) failed to protect cattle from generalized clinical disease following experimental FMDV Asia 1 infection. Our results showed that infection induced higher and prolonged serum antibody titres indicating antigen mass is important for optimal immune response. Experimental FMDV infection induced significant secretory antibody (mucosal) response in cattle. Though, there was no difference in the serum antibody response between the cattle that developed generalized infection (unprotected) and those with only localized infection (protected), secretory antibody response differed, wherein the unprotected cattle had higher secretory response than protected cattle. Thus, FMDV Asia 1 infection stimulates a similar serum antibody response and a unique secretory antibody response among the infected cattle.

The mucosal immune response to foot-and-mouth disease virus (FMDV) type Asia 1 was examined in experimentally infected cattle by assaying antibodies by the virus-neutralizing test (VNT) and IgA ELISA in two secretory fluids, oesophageal pharyngeal fluid (OPF) and oro-nasal fluid (ONF). Out of 17 animals infected by the intradermo-lingual route, 12 became persistently infected (carriers), as defined by positive antigen capture RT-PCR reactions for FMDV RNA in OPF samples collected at 28 days or later after exposure. This proportion of carriers (71%) with FMDV Asia 1 is comparable to other serotypes of the virus. When the two groups were examined, the carriers and non-carriers showed no difference in the serum antibody titre until the end of the experiment at 182 days post-infection (DPI). However, despite an initial similarity significantly higher neutralizing antibody titres and FMDV-specific IgA response were detected among the carriers than the non-carriers in both of the secretory fluids. The response was higher and more stable in ONF compared to OPF. Thus, mucosal antibody assays have the potential to be used as a means of differentiating carrier from non-carrier cattle. Furthermore, the findings are consistent with the higher mucosal antibody response in carriers being an effect of persistent infection rather than the cause.

Foot-and-mouth disease (FMD) is one of the most contagious diseases affecting wide range of host species with variable severity and decreased productivity. The present study was undertaken to compare the clinical and leucocytic changes in indigenous Indian cattle and buffaloes experimentally infected with FMD virus (FMDV) Asia 1. A mild type of disease was observed in the cattle, more so in buffaloes infected with FMDV. Difference in terms of type, site and healing of lesion was observed between cattle and buffaloes. Foot lesions were more common than tongue in buffaloes, which were mainly evident in bulb of the heel in contrast to interdigital foot lesions in cattle. Further, FMDV infection induced a transient moderate leucopenia with lymphopenia in both cattle and buffaloes, but monocyte levels diverged. Relationship between the raised body temperature, leucocytic changes and lesion development was observed. Microscopic changes were observed in the keratinized epithelium of tongue and foot. The findings of the present study indicated the need to investigate the early leucocytic changes in cattle and buffaloes in depth for better understanding of the disease process.

Haemonchosis is a very common disease in small ruminants caused by H. contortus, a blood sucking parasite causing anaemia that may be fatal particularly to young animals. Therefore, detection of the infection during prepatent period is important for early treatment. Excretory-Secretory (ES) protein of H. contortus was purified through immunoaffinity chromatography. Dot -ELISA was performed with crude ES antigen as well as immunoaffinity purified fraction (F-1) with experimental and natural sera of sheep infected with H. contortus. Solid dot formation took place with 4 day, 1, 2 and 3 weeks post infection sera. Dot formation did not take place with negative control serum and uninfected control animal serum. When crude ES antigens was reacted to natural sheep sera having H. contortus infection, 60% sera samples showed solid dot formation whereas in F-1 fraction 75% of the sera samples showed solid dot indicating purified fraction was a more potent antigen. Crude ES and F-1 were also fractionated through SDS-PAGE. ES antigen revealed polypeptides in the range of 10 to 200 kDa of which 26, 32, 60 and 120 kDa were found more prominent. F-1 fraction on SDS-PAGE analysis revealed only four polypeptides of 26, 32, 60, and 120 of which 60 and 120 kDa were found to be most prominent. Results indicate that the purified fraction of ES antigen may be utilized for early diagnosis of haemonchosis. Further studies on cross antigenicity of this fraction with other nematode and trematode needs to be conducted.

The phylogenetic relationships of five isolates of Pasteurella multocida serotype B:2 belonging to buffalo, cattle, pig, sheep and goat were investigated by comparative sequence analysis of 16S rRNA gene. The 1468bp fragment of 16S rRNA gene sequence comparison showed that the isolates of cattle (PM75), pig (PM49) and sheep (PM82) shared 99.9% homology with the buffalo isolate (vaccine strain P52) whereas, the goat isolate (PM86) shared 99.8% homology with the vaccine strain. The 16S rRNA gene sequences of these isolates were also found monophyletic with type B reference strain NCTC 10323 of P. multocida subsp. multocida. The present study indicated the close relationships of haemorrhagic septicaemia causing P. multocida serotype B:2 isolates of buffalo and cattle with other uncommon hosts (pig, sheep and goat).

Comparative efficacy of an IS900 tissue PCR, bacterial culture, johnin, agar-gel immunodiffusion (AGID) and absorbed-ELISA tests was investigated in 43 goats naturally infected with paratuberculosis. On histological examination, tissue sections from all animals showed typical granulomatous inflammatory changes. The lesions were classified as multibacillary (MB) (n=30), which had diffuse granulomatous lesions with abundant acid-fast bacilli (AFB), and paucibacillary (PB) (n=13), which had focal or multifocal granulomatous lesions with few AFB. The sensitivities of johnin test, tissue culture, faecal culture, tissue PCR, AGID and ELISA were 68% (17/25), 100% (30/30), 84.6% (22/26), 100% (30/30), 96.2% (25/26) and 100% (26/26) in MB goats, and 88.8 (8/9), 46.1% (8/13), 40% (4/10), 61.5% (8/13), 50% (5/10), and 70% (7/10) in PB goats, respectively. Except for the johnin test, which showed higher sensitivity in PB goats, all other tests displayed significantly higher sensitivities in MB goats. The results indicate the usefulness of tissue PCR, culture and serological tests in the diagnosis of clinically affected paratuberculous goats, especially with multibacillary pathology.

Applicability of polymerase chain reaction (PCR) assay to detect Pasteurella multocida in experimentally infected embryonated chicken egg was assessed in the present study. PCR assay rapidly and specifically detected the genome of P. multocida in amniotic fluid, allantoic fluid and homogenates of infected embryo and its membranes. The sensitivity of detection was as low as 20 bacterial cells/ml of allantoic or amniotic fluids. Detection of P. multocida in dead embryos by PCR was possible up to 6 and 30 days or more following storage of dead embryos at 37 degrees C, and at 4 degrees C as well as at -20 degrees C, respectively. The study revealed that PCR assays could be employed directly for detection and confirmation of P. multocida infection in experimentally infected chicken embryos.

The efficacy of bacterial culture and IS900-specific polymerase chain reaction (PCR) was compared for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) from the intestinal and mesenteric lymph node tissues of water buffaloes (Bubalus bubalis) showing lesions of paratuberculosis (Johne's disease). Out of 20 (4.9%) animals showing histological lesions suggestive of paratuberculosis, 14 (70%) and 6 (30%) were positive in the PCR and bacterial culture, respectively. The results of this study suggested that PCR was more sensitive than bacterial culture in detection of subclinical paratuberculosis in water buffaloes. The bacterial concentration from large amount of tissues by differential and density gradient centrifugation method was found to facilitate the diagnosis by smear examination and PCR. The specificity of the PCR was confirmed by the product size and restriction digestion pattern of the amplicons. The sequence analysis of the amplified products (626bp of IS900 gene) from buffalo strain showed more than 97% homology with the published sequences.