Dual parametric sensors for highly sensitive nucleic acid detection

The primary focus of this research work was on the design and development of a molecular scale (nano-scale) capacitive sensing mechanism for the highly sensitive and label-free detection of Nucleic Acid hybridization. These novel capacitive sensors with nano-scale electrode spacing offer solutions to many problems suffered by the conventional signal transduction mechanisms, thereby immensely improving the sensitivity of the biomolecular detection processes. Reducing the separation between the capacitive electrodes to the same scale as the Debye length of the sample solution, results in the overlapping of the electrical double layers of the two electrodes, thereby confining them to occupy a major fraction of the dielectric volume. This decreases the potential drop across the electrodes and thus dielectric measurements at low frequencies are made possible. The dielectric properties during hybridization reaction were measured using 10- mer nucleotide sequences. A 30-40% change in relative permittivity (capacitance) was observed due to DNA hybridization at 10Hz, which is much more sensitive than the previously reposted detection measurements (2-8% signal change). In parallel to the above work, a second label-free sensing mechanism based on field effect capacitive sensors with Metal-Oxide-Semiconductor (MOS) structure has been developed and its ability to provide real-time monitoring of oligonucleotide immobilization and hybridization events are studied. The immobilization of probe oligomers on the sensor surface and their hybridization with the target oligomers of complimentary sequences has produced significant shifts (140mV and 73mV respectively) in the Capacitance-Voltage characteristics measured across the device. In an attempt to utilize the individual merits of the nano-scale electrochemical capacitive sensor and the field effect MOS capacitive structure, a novel dual parametric sensing architecture comprising of both these transducing elements on a single sensor is designed. The detection scheme based on the combined analysis of the two parameters- Dielectric property and intrinsic molecular charge- of Nucleic acid molecules has found to reveal complimentary information of significance about the analyte-probe interactions. As a separate experiment the applications and promises of a novel technique of enhancing the speed and selectivity of the molecular detection processes by the application of an external electric field of precisely controlled intensity was studied. Experiments were conducted with 10-mer sequences and proved the feasibility of this technique in inducing in providing a faster and selective immobilization and hybridization reactions. The research work in this direction has been in collaboration with the Rational Affinity Devices, LLC, a New Jersey based corporation. The above mentioned biosensing mechanisms and detection techniques have the advantage of simplifying the readout and increasing the speed and ease of nucleic acid assays, which is especially desirable for characterizing infectious agents, scoring sequence polymorphism and genotypes, and measuring mRNA or miRNA levels during expression profiling. Once fully optimized and well assembled they have great potential to be developed in to a commercial full-scale biosensor capable of providing high-value diagnostic testing at the point of patient care places

Automated tight Lyapunov analysis for first-order methods

We present a methodology for establishing the existence of quadratic Lyapunov inequalities for a wide range of first-order methods used to solve convex optimization problems. In particular, we consider i) classes of optimization problems of finite-sum form with (possibly strongly) convex and possibly smooth functional components, ii) first-order methods that can be written as a linear system on state-space form in feedback interconnection with the subdifferentials of the functional components of the objective function, and iii) quadratic Lyapunov inequalities that can be used to draw convergence conclusions. We provide a necessary and sufficient condition for the existence of a quadratic Lyapunov inequality that amounts to solving a small-sized semidefinite program. We showcase our methodology on several first-order methods that fit the framework. Most notably, our methodology allows us to significantly extend the region of parameter choices that allow for duality gap convergence in the Chambolle-Pock method when the linear operator is the identity mapping

The Mare Reproductive Loss Syndrome and the Eastern Tent Caterpillar: A Toxicokinetic/Statistical Analysis With Clinical, Epidemiologic, and Mechanistic Implications

During 2001, central Kentucky experienced acute transient epidemics of early and late fetal losses, pericarditis, and unilateral endophthalmitis, collectively referred to as mare reproductive loss syndrome (MRLS). A toxicokinetic/statistical analysis of experimental and field MRLS data was conducted using accelerated failure time (AFT) analysis of abortions following administration of Eastern tent caterpillars (ETCs; 100 or 50 g/day or 100 g of irradiated caterpillars/day) to late-term pregnant mares. In addition, 2001 late-term fetal loss field data were used in the analysis. Experimental data were fitted by AFT analysis at a high (P \u3c .0001) significance. Times to first abortion (“lag time”) and abortion rates were dose dependent. Lag times decreased and abortion rates increased exponentially with dose. Calculated dose × response data curves allow interpretation of abortion data in terms of “intubated ETC equivalents.” Analysis suggested that field exposure to ETCs in 2001 in central Kentucky commenced on approximately April 27, was initially equivalent to approximately 5 g of intubated ETCs/day, and increased to approximately 30 g/day at the outbreak peak. This analysis accounts for many aspects of the epidemiology, clinical presentations, and manifestations of MRLS. It allows quantitative interpretation of experimental and field MRLS data and has implications for the basic mechanisms underlying MRLS. The results support suggestions that MRLS is caused by exposure to or ingestion of ETCs. The results also show that high levels of ETC exposure produce intense, focused outbreaks of MRLS, closely linked in time and place to dispersing ETCs, as occurred in central Kentucky in 2001. With less intense exposure, lag time is longer and abortions tend to spread out over time and may occur out of phase with ETC exposure, obscuring both diagnosis of this syndrome and the role of the caterpillars

Development of high-affinity nanobodies specific for NaV1.4 and NaV1.5 voltage-gated sodium channel isoforms

Voltage-gated sodium channels, NaVs, are responsible for the rapid rise of action potentials in excitable tissues. NaV channel mutations have been implicated in several human genetic diseases, such as hypokalemic periodic paralysis, myotonia, and long-QT and Brugada syndromes. Here, we generated high-affinity anti-NaV nanobodies (Nbs), Nb17 and Nb82, that recognize the NaV1.4 (skeletal muscle) and NaV1.5 (cardiac muscle) channel isoforms. These Nbs were raised in llama (Lama glama) and selected from a phage display library for high affinity to the C-terminal (CT) region of NaV1.4. The Nbs were expressed in Escherichia coli, purified, and bio-physically characterized. Development of high-affinity Nbs specifically targeting a given human NaV isoform has been challenging because they usually show undesired cross-reactivity for different NaV isoforms. Our results show, however, that Nb17 and Nb82 recognize the CTNaV1.4 or CTNaV1.5 over other CTNav isoforms. Kinetic experiments by biolayer interferometry determined that Nb17 and Nb82 bind to the CTNaV1.4 and CTNaV1.5 with high affinity (KD ~ 40-60 nM). In addition, as proof of concept, we show that Nb82 could detect NaV1.4 and NaV1.5 channels in mammalian cells and tissues by Western blot. Furthermore, human embryonic kidney cells expressing holo NaV1.5 channels demonstrated a robust FRET-binding efficiency for Nb17 and Nb82. Our work lays the foundation for developing Nbs as anti-NaV reagents to capture NaVs from cell lysates and as molecular visualization agents for NaVs.Fil: Srinivasan, Lakshmi. University Johns Hopkins; Estados UnidosFil: Alzogaray, Vanina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Selvakumar, Dakshnamurthy. Fortébio; Estados UnidosFil: Nathan, Sara. University Johns Hopkins; Estados UnidosFil: Yoder, Jesse B.. University Johns Hopkins; Estados UnidosFil: Wright, Katharine M.. University Johns Hopkins; Estados UnidosFil: Klinke, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Nwafor, Justin N.. University Johns Hopkins; Estados UnidosFil: Labanda, María Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Goldbaum, Fernando Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Schön, Arne. University Johns Hopkins; Estados UnidosFil: Freire, Ernesto. University Johns Hopkins; Estados UnidosFil: Tomaselli, Gordon F.. University Johns Hopkins; Estados UnidosFil: Amzel, León Mario. University Johns Hopkins; Estados UnidosFil: Ben-Johny, Manu. Columbia University; Estados UnidosFil: Gabelli, Sandra. University Johns Hopkins; Estados Unido

Wwox Deletion in Mouse B Cells Leads to Genomic Instability, Neoplastic Transformation, and Monoclonal Gammopathies

WWOX (WW domain containing oxidoreductase) expression loss is common in various cancers and characteristic of poor prognosis. Deletions, translocations, and loss of expression affecting the WWOX gene are a common feature of various B cell neoplasms such as certain B cell lymphomas and multiple myeloma. However, the role of this common abnormality in B cell tumor initiation and/or progression has not been defined. In this study, we conditionally deleted Wwox early in B cell development by means of breeding Cd19-Cre transgenic mice crossed to Wwox floxed mice (Cd19 Wwox KO). We observed a significant reduced survival in Cd19 Wwox KO mice and the development of B cell neoplasms including B cell lymphomas, plasma cell neoplasias characterized by increased numbers of CD138+ populations as well as monoclonal gammopathies detected by serum protein electrophoresis. To investigate whether Wwox loss could play a role in genomic instability, we analyzed DNA repair functions during immunoglobulin class switch joining between DNA segments in antibody genes. While class switch recombination (CSR) was only slightly impaired, Wwox deficiency resulted in a dramatic shift of double strand break (DSB) repair from normal classical-NHEJ toward the microhomology-mediated alternative-NHEJ pathway, a pathway associated with chromosome translocations and genome instability. Consistent with this, Wwox deficiency resulted in a marked increase of spontaneous translocations during CSR. This work defines for the first time a role for Wwox for maintaining B cell genome stability during a process that can promote neoplastic transformation and monoclonal gammopathies.Facultad de Ciencias MédicasCentro de Investigaciones Inmunológicas Básicas y Aplicada

Cyclin-dependent kinase 12 is a drug target for visceral leishmaniasis

Visceral leishmaniasis causes considerable mortality and morbidity in many parts of the world. There is an urgent need for the development of new, effective treatments for this disease. Here we describe the development of an anti-leishmanial drug-like chemical series based on a pyrazolopyrimidine scaffold. The leading compound from this series (7, DDD853651/GSK3186899) is efficacious in a mouse model of visceral leishmaniasis, has suitable physicochemical, pharmacokinetic and toxicological properties for further development, and has been declared a preclinical candidate. Detailed mode-of-action studies indicate that compounds from this series act principally by inhibiting the parasite cdc-2-related kinase 12 (CRK12), thus defining a druggable target for visceral leishmaniasis

Current gaps in sepsis immunology: new opportunities for translational research

Increasing evidence supports a central role of the immune system in sepsis, but the current view of how sepsis affects immunity, and vice versa, is still rudimentary. The European Group on Immunology of Sepsis has identified major gaps that should be addressed with high priority, such as understanding how immunological alterations predispose to sepsis, key aspects of the immunopathological events during sepsis, and the long-term consequences of sepsis on patient's immunity. We discuss major unmet topics in those three categories, including the role of key immune cells, the cause of lymphopenia, organ-specific immunology, the dynamics of sepsis-associated immunological alterations, the role of the microbiome, the standardisation of immunological tests, the development of better animal models, and the opportunities offered by immunotherapy. Addressing these gaps should help us to better understand sepsis physiopathology, offering translational opportunities to improve its prevention, diagnosis, and care

Complementing compost with biochar for agriculture, soil remediation and climate mitigation

We are racing to manage a phenomenally increasing volume of organic wastes from urban, industrial and agricultural entities. Composting is one of the preferred ways to convert biodegradable wastes into nutrient-rich soil conditioners. The age-old technique of composting process is being improved with innovative scientific means. Biochar, a widely studied soil amendment, is a carbonaceous material that can hold nutrients from endogenic/exogenic sources. Biochar-compost, a biochar-complemented compost, may provide a wide range of benefits expected from both materials. Compost and biochar can improve physicochemical and microbiological attributes of soils by supplying labile and stable carbons, and nutrients. Compost may also supply beneficial microbes. This means biochar-compost is a synergic soil amendment that can improve soil quality, increase crop production, and remediate contaminated soils. Having stable carbon, large reactive surface with nutrient loads, biochar can interact widely with organic biomass and modify physicochemical and-microbial states during a composting process while making biochar-compost. Production and application methods of biochar, compost and biochar-compost are covered for agricultural and contaminated soils. Metal and organic contaminations are also discussed. A case study on making and field-testing a mineral-enhanced biochar and a biochar-compost to improve rice yield, is presented at the end