Transit time estimation of drying springs in Uttarakhand region using environmental tritium concentration.

Many parts of the Uttarakhand state, situated in the hilly terrain of the Himalayan region of India are facing acute water crisis due to the drying up of the perennial springs which are the only source of potable water in those regions. Tritium (3H), the radioactive isotope of hydrogen (half-life of 12.32 years) and also a part of water molecule (in the form of HTO) acts as a very useful tracer in estimating the transit time of the hydrological systems. Tritium concentrations of three springs (S-1, S-2 and S-3) were monitored consecutively for three years (2017-2019) to better constrain the transit time estimation. The tritium concentrations of the springs are found to vary between 3.66 and 4.15 TU. All the springs show gradual decrease in tritium concentration with the passage of time indicating the diminishing percentage of freshly recharged modern water component. Among various lumped parameter models, the piston-flow model (PFM), exponential mixing model (EMM), exponential piston-flow model (EPM) and partial exponential mixing model (PEM) have been employed in this study. The historical record of weighted mean concentration of tritium in precipitation available for the Uttarakhand region is taken as input function in the modelling procedure. The application of various LPMs (PFM, EMM, EPM and PEM) indicates that the transit time of the S-1 spring ranges from 1.26 to 1.46 years whereas for S-2 spring, the transit time is found to vary from 5 months to 1.1 years. The MTT of S-3 spring ranges from 5 months to 11 months. The relatively short residence time of these springs indicates the actively recharged system. The estimation of accurate transit time is thus very crucial for understanding the renewability of the spring water systems.

TP5: A Novel Therapeutic Approach Targeting Aberrant and Hyperactive CDK5/p25 for the Treatment of Colorectal Carcinoma.

Colorectal carcinoma (CRC) is a prevalent cancer worldwide with a high mortality rate. Evidence suggests that increased expression of Cyclin-dependent kinase 5 (CDK5) contributes to cancer progression, making it a promising target for treatment. This study examined the efficacy of selectively inhibiting CDK5 in colorectal carcinoma using TP5, a small peptide that selectively inhibits the aberrant and hyperactive CDK5/p25 complex while preserving physiological CDK5/p35 functions. We analyzed TP5's impact on CDK5 activity, cell survival, apoptosis, the cell cycle, DNA damage, ATM phosphorylation, and reactive oxygen species (ROS) signaling in mitochondria, in CRC cell lines, both alone and in combination with chemotherapy. We also assessed TP5's efficacy on a xenograft mouse model with HCT116 cells. Our results showed that TP5 decreased CDK5 activity, impaired cell viability and colony formation, induced apoptosis, increased DNA damage, and led to the G1 phase arrest of cell cycle progression. In combination with irinotecan, TP5 demonstrated a synergy by leading to the accumulation of DNA damage, increasing the γH2A.X foci number, and inhibiting G2/M arrest induced by Sn38 treatment. TP5 alone or in combination with irinotecan increased mitochondrial ROS levels and inhibited tumor growth, prolonging mouse survival in the CRC xenograft animal model. These results suggest that TP5, either alone or in combination with irinotecan, is a promising therapeutic option for colorectal carcinoma.

Investigation of flow dynamics of molten glass in different solar glass production units using radiotracer method.

A radiotracer investigation was previously carried out to characterize the flow of the molten glass and to identify the cause of poor quality of the glass sheets produced in an industrial solar glass production unit (SGPU-1) (Pant et al., 2016). Based on the investigations, several flow abnormalities were identified and the design of the unit was modified to improve the quality of the product and meet the product specifications. Subsequently, the radiotracer investigation was repeated in the modified unit (SGPU-1m). The results of the study showed that the dead volume and homogenization time in the modified unit were significantly reduced with improvement in mixing of the molten glass as compared to the SGPU-1. Based on the results of the two investigations, a new glass production unit (SGPU-2) with enhanced capacity was designed, fabricated and commissioned. The radiotracer investigation was repeated in the newly designed unit with an objective to evaluate and validate its design. The results indicated that the performance of SGPU-2 was as per the design criteria and the quality of the glass sheets produced was as per the desired specifications.

Cyclin-Dependent Kinase 5 Regulates cPLA2 Activity and Neuroinflammation in Parkinson's Disease.

Hyperactivation of cyclin-dependent kinase 5 (Cdk5) by p25, contributes to neuroinflammation causing neurodegeneration in Parkinson's disease (PD) and Alzheimer's disease. However, the mechanism by which Cdk5 induces neuroinflammation in the PD brain is largely unexplored. Here, we show that Cdk5 phosphorylates cytosolic phospholipase A2 (cPLA2) at Thr-268 and Ser-505 sites lead to its activation and generation of eicosanoid products. Mutational studies using site-directed mutagenesis and molecular simulations show that the architecture of the protein changes on each single-point mutation. Interestingly, double mutations also led to a severe decline in the activity of cPLA2 and to the disruption of its translocation to the plasma membrane. Further, the brain lysates of transgenic PD mouse models show hyperactivation of Cdk5, resulting in enhanced phosphorylation of Thr-268 and Ser-505 of cPLA2 and its heightened activity, confirming the findings observed in the cell culture model of PD. These phosphorylation sites of cPLA2 and Cdk5 could be explored as the future therapeutic targets against neuroinflammation in PD. Further, conjoint transcriptomic analysis of the publicly available human PD datasets strengthens the hypothesis that genes of the arachidonic acid, prostaglandin synthesis, and inflammatory pathways are significantly upregulated in the case of PD patients compared with that of healthy control subjects.

Investigation of aqueous phase dynamics in a uranium stripping unit using radioactive tracer.

Mixer-setters units are widely used in uranium purification processes. For efficient operations of mixer-settler units, it is essential to measure the hydrodynamics parameters of the different phases involved. The residence time distribution (RTD) measurement is commonly used method to estimate the hydrodynamics parameters of process reactors. In the present study, RTD of the aqueous phase was measured in different stages mixer-settler unit (mixers, settlers and mixer-settler units) used for stripping operation using Iodine-131 as a radiotracer. For the RTD measurements, radiotracer was injected as an impulse in aqueous phase feed line and its movement was monitored at different locations in the stripping unit using NaI(Tl) detectors. The mean residence times (MRTs) of the aqueous phase were estimated from measured RTD curves. For quantification of the degree of mixing, suitable flow models were proposed based on design and nature of the measured RTD curves and subsequently used for simulation. Based on the results of the RTD study, the mixing of aqueous phase was characterized and design of the stripping unit and its sub-units were validated. The optimum conditions were identified for efficient for the operation of the stripping unit.

Computational Study of the Allosteric Effects of p5 on CDK5-p25 Hyperactivity as Alternative Inhibitory Mechanisms in Neurodegeneration.

The cyclin-dependent kinase (CDK5) forms a stable complex with its activator p25, leading to the hyperphosphorylation of tau proteins and to the formation of plaques and tangles that are considered to be one of the typical causes of Alzheimer's disease (AD). Hence, the pathological CDK5-p25 complex is a promising therapeutic target for AD. Small peptides, obtained from the truncation of CDK5 physiological activator p35, have shown promise in inhibiting the pathological complex effectively while also crossing the blood-brain barrier. One such small 24-residue peptide, p5, has shown selective inhibition toward the pathological complex in vivo. Our previous research focused on the characterization of a computationally predicted CDK5-p5 binding mode and of its pharmacophore, which was consistent with competitive inhibition. In continuation of our previous work, herein, we investigate four additional binding modes to explore other possible mechanisms of interaction between CDK5 and p5. The quantitative description of the pharmacophore is consistent with both competitive and allosteric p5-induced inhibition mechanisms of CDK5-p25 pathology. The gained insights can direct further in vivo/in vitro tests and help design small peptides, linear or cyclic, or peptidomimetic compounds as adjuvants of orthosteric inhibitors or as part of a cocktail of drugs with enhanced effectiveness and lower side effects.

Preparation and characterization of cobalt-60 glass microspheres for radioactive particle tracking applications.

The current work describes development and optimization of a process for preparation of cobalt-60 glass microspheres. These microspheres have potential for applications in radioactive particle tracking (RPT) studies in multiphase flow systems. In the first step of preparation, soda lime glass containing 5-10 wt% cobalt oxide was produced through melt-quench method. Subsequently, cobalt glass microspheres (CMSs) were prepared by microwave heating of tiny glass grains in presence of graphite. In the final step, radioactive cobalt-60 microspheres (RMSs) were produced by neutron irradiation of the CMSs in a nuclear reactor. The CMSs were characterized for surface morphology, elemental composition, homogeneity, crystalinity using SEM, EDX and XRD, respectively. The thermal behaviour of the microspheres was characterized by TG and DSC analysis. The size distribution of CMSs analyzed by SEM was found to be in the range 500-2000 µm. The preparation step was optimized to produce adequate activity in a single microsphere, so that they can be utilized for RPT applications.

Protocols for Characterization of Cdk5 Kinase Activity.

Cyclin-dependent kinases (Cdks) are generally known to be involved in controlling the cell cycle, but Cdk5 is a unique member of this protein family for being most active in post-mitotic neurons. Cdk5 is developmentally important in regulating neuronal migration, neurite outgrowth, and axon guidance. Cdk5 is enriched in synaptic membranes and is known to modulate synaptic activity. Postnatally, Cdk5 can also affect neuronal processes such as dopaminergic signaling and pain sensitivity. Dysregulated Cdk5, in contrast, has been linked to neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). Despite primarily being implicated in neuronal development and activity, Cdk5 has lately been linked to non-neuronal functions including cancer cell growth, immune responses, and diabetes. Since Cdk5 activity is tightly regulated, a method for measuring its kinase activity is needed to fully understand the precise role of Cdk5 in developmental and disease processes. This article includes methods for detecting Cdk5 kinase activity in cultured cells or tissues, identifying new substrates, and screening for new kinase inhibitors. Furthermore, since Cdk5 shares homology and substrate specificity with Cdk1 and Cdk2, the Cdk5 kinase assay can be used, with modification, to measure the activity of other Cdks as well. © 2021 Wiley Periodicals LLC. This article has been contributed to by US Government employees and their work is in the public domain in the USA. Basic Protocol 1: Measuring Cdk5 activity from protein lysates Support Protocol 1: Immunoprecipitation of Cdk5 using Dynabeads Alternate Protocol: Non-radioactive protocols to measure Cdk5 kinase activity Support Protocol 2: Western blot analysis for the detection of Cdk5, p35, and p39 Support Protocol 3: Immunodetection analysis for Cdk5, p35, and p39 Support Protocol 4: Genetically engineered mice (+ and - controls) Basic Protocol 2: Identifying new Cdk5 substrates and kinase inhibitors.

Residence time distribution study in a pilot-scale liquid phase catalytic exchange (LPCE) column packed with a mixture of hydrophobic and hydrophilic catalysts.

Residence time distribution (RTD) measurements were carried out in a packed bed column designed for exchange of hydrogen isotopes. The main objective of the study was to characterize the liquid phase mixing under various processes and operating conditions. The packed bed was composed of a mixture of two different types of catalytic packing materials, i.e., a hydrophobic material and a hydrophilic material. Technitium-99m (99mTc) as sodium pertechnetate was used as a radiotracer for RTD measurements. From the measured RTD curves, mean residence times (MRTs), liquid holdup and degree of mixing of liquid phase were evaluated. An axial dispersion model exchange with stagnant zones was used to simulate the measured RTD curves. The results of model simulation showed that volume fraction of hydrophobic to hydrophilic packing and gas/liquid superficial velocities affect the liquid holdup, bed pressure drop and liquid phase dispersion/mixing characteristics. The results of the present study will help to screen packing, optimize the volume of the packing fractions, design and construct the catalyst and optimize the operating conditions for scale up of the isotope exchange process.

Therapeutic effects of TP5, a Cdk5/p25 inhibitor, in in vitro and in vivo models of Parkinson's disease.

Parkinson's Disease (PD) is a chronic progressive neurodegenerative disease. Current treatments for PD are symptomatic and only increase striatal dopamine levels. Proactive neuroprotective approaches that slow the progression of PD and maintain appropriate dopamine neuron populations are needed to treat the disease. One suggested mechanism contributing to the pathology of PD involves the binding of cyclin-dependent kinase 5 (Cdk5) to p25, creating a hyperactivated complex to induce cell death. The objective of this study is to investigate the neuroprotective and neurorestorative properties of Truncated Peptide 5 (TP5), a derivative of the p35 activator involved in Cdk5 regulation, via the inhibition of Cdk5/p25 complex function. SH-SY5Y cell line and the nematode Caenorhabditis elegans were exposed to paraquat (PQ), an oxidative stressor, to induce Parkinsonian phenotypes. TP5 was administered prior to PQ exposure to determine its neuroprotective effects and, in further experiments, after PQ exposure to examine its neurorestorative effects. In the SH-SY5Y cell line, TP5 was found to have neuroprotective effects using a cell viability assay and demonstrated neuroprotective and neurorestorative effects in C. elegans by examining dopaminergic neurons and dopamine-dependent behaviour. TP5 decreased elevated Cdk5 activation in worms that were exposed to PQ. TP5's inhibition of Cdk5/p25 hyperactivity led to the protection of dopamine neurons in these PD models. This suggests that TP5 can act as a potential therapeutic drug towards PD.

Methods of preparation of microparticles for radioactive particle tracking experiments.

Radioactive particle tracking (RPT) technique is a relatively newer technique for the characterization of flow of process materials (liquids, solids) in laboratory- and pilot-scale industrial systems. The technique uses a single particle labelled with a suitable radioisotope having similar physical properties to that of the bulk of the process material. The preparation of a representative radioactive microparticle is a challenging task in the implementation of the technique. There are no standard methods available for the preparation of radioactive microparticles. This paper discusses some of the methods of preparation of radioactive microparticles for RPT studies. A few examples of RPT applications using the prepared microparticles are also discussed.

Hydrodynamic modeling and radiotracer investigations in Kolkata Port Trust, Kolkata, West Bengal, India.

In this study, hydrodynamic modeling and radiotracer investigations were conducted in Kolkata Port Trust, Kolkata, West Bengal, India to investigate the dispersion of sediments and to evaluate the suitability of three different dumping sites (Upper Auckland, Lower Auckland and Eden) for dredged sediments along a navigation channel. Scandium-46 (148-240 GBq) comprising scandium glass powder with a particle size distribution ranging from 75-100 µm was used as a radiotracer. The radiotracer was injected onto the seabed at the selected site using a remotely operated injection system and its movement on the seabed was tracked over a period of three months using waterproof NaI(Tl) scintillation detector. The tracer concentration curves were plotted on a site map of the area for different trackings and isocount contours were plotted. Qualitative information was obtained from the isocount contours such as the general direction of movement and spread of the radiotracer. The isocount contours showed that the sediment predominantly moved away from the navigation channel. From the isocount, a transport diagram was plotted based on isocount counter and sediment transport parameters such as the general direction of movement by sediments on the seabed, transport velocity, transport thickness and bed load movement rate were determined for all the sites. The dispersion patterns obtained for the sediments by modeling were compared with the experimental results and they were found to be similar. Thus, the model results were validated and the proposed sites were found to be suitable for dumping the dredged material because the sediments did not return to the navigation channel.

TP5, a Peptide Inhibitor of Aberrant and Hyperactive CDK5/p25: A Novel Therapeutic Approach against Glioblastoma.

We examined the efficacy of selective inhibition of cyclin-dependent kinase 5 (CDK5) in glioblastoma by TP5. We analyzed its impact in vitro on CDK5 expression and activity, cell survival, apoptosis and cell cycle. DNA damage was analyzed using the expression of γH2A.X and phosphorylated ATM. Its tolerance and efficacy were assessed on in vivo xenograft mouse models. We showed that TP5 decreased the activity but not the expression of CDK5 and p35. TP5 alone impaired cell viability and colony formation of glioblastoma cell lines and induced apoptosis. TP5 increased DNA damage by inhibiting the phosphorylation of ATM, leading to G1 arrest. Whereas CDK5 activity is increased by DNA-damaging agents such as temozolomide and irradiation, TP5 was synergistic with either temozolomide or irradiation due to an accumulation of DNA damage. Concomitant use of TP5 and either temozolomide or irradiation reduced the phosphorylation of ATM, increased DNA damage, and inhibited the G2/M arrest induced by temozolomide or irradiation. TP5 alone suppressed the tumor growth of orthotopic glioblastoma mouse model. The treatment was well tolerated. Finally, alone or in association with irradiation or temozolomide, TP5 prolonged mouse survival. TP5 alone or in association with temozolomide and radiotherapy is a promising therapeutic option for glioblastoma.

Rapid, quantitative therapeutic screening for Alzheimer's enzymes enabled by optimal signal transduction with transistors.

We show that commercially sourced n-channel silicon field-effect transistors (nFETs) operating above their threshold voltage with closed loop feedback to maintain a constant channel current allow a pH readout resolution of (7.2 ± 0.3) × 10-3 at a bandwidth of 10 Hz, or ≈3-fold better than the open loop operation commonly employed by integrated ion-sensitive field-effect transistors (ISFETs). We leveraged the improved nFET performance to measure the change in solution pH arising from the activity of a pathological form of the kinase Cdk5, an enzyme implicated in Alzheimer's disease, and showed quantitative agreement with previous measurements. The improved pH resolution was realized while the devices were operated in a remote sensing configuration with the pH sensing element off-chip and connected electrically to the FET gate terminal. We compared these results with those measured by using a custom-built dual-gate 2D field-effect transistor (dg2DFET) fabricated with 2D semi-conducting MoS2 channels and a signal amplification of 8. Under identical solution conditions the nFET performance approached the dg2DFETs pH resolution of (3.9 ± 0.7) × 10-3. Finally, using the nFETs, we demonstrated the effectiveness of a custom polypeptide, p5, as a therapeutic agent in restoring the function of Cdk5. We expect that the straight-forward modifications to commercially sourced nFETs demonstrated here will lower the barrier to widespread adoption of these remote-gate devices and enable sensitive bioanalytical measurements for high throughput screening in drug discovery and precision medicine applications.

Investigation of flow dynamics of primary coolant in a delay tank of a swimming pool-type nuclear reactor using radiotracer technique.

Radiotracer investigations were carried out for tracing primary coolant in a delay tank of a swimming pool type nuclear reactor. The delay tank was designed to provide a certain delay or residence time to the primary coolant so that the short-lived radioisotopes such as (nitrogen-16 and oxygen-19) decay to a safer level before exiting from the delay tank. However, soon after commissioning of the reactor, the radiation levels at the exit of the reactor core and delay tank, in the working area were found to be higher than the permissible levels. Therefore, the main objectives of the investigations were to measure breakthrough and residence times and, to investigate flow dynamics of the coolant within the tank. Residence time distributions (RTDs) of the coolant were measured in the delay tank using technetium-99m as sodium pertechnatate as a radiotracer. The breakthrough time (BTT) and mean residence time (MRT) were determined from the measured RTD and the same were found to be inadequate to allow the decay of short-lived radioisotopes to the permissible levels. Axial dispersion model with two parallel flow streams was used to simulate the measured RTD curves. Results of the model simulation indicated bypassing of the coolant. Based on the results of the radiotracer investigations, necessary modifications were carried out in the design of the tank. After implementing the modifications, the radiotracer experiments were repeated and, the BTT and the MRT were found to increase sufficient enough to allow decay of the produced radioisotopes and thus to reduce the radiation levels at the exit of the delay tank and in the working area to the safer and permissible levels.

Investigation of flow dynamics of porous clinkers in a ball mill using technitium-99m as a radiotracer.

A radiotracer investigation was carried out in a ball mill of a cement plant in Kenya. Residence time distribution (RTD) of raw feed to the mill was measured using Technetium-99m adsorbed on the clinkers as a radiotracer. From the measured RTDs, solid holdup and mean residence times (MRTs) in the ball mill and associated separator were determined. The measured RTDs were modelled using axial dispersion model (ADM) and tank-in-series model both connected with a plug flow component in series. The results of the modelling indicated significant degree of backmixing within the ball mill and no axial mixing in the separator.

Quantum capacitance-limited MoS2 biosensors enable remote label-free enzyme measurements.

We have demonstrated atomically thin, quantum capacitance-limited, field-effect transistors (FETs) that enable the detection of pH changes with 75-fold higher sensitivity (≈4.4 V per pH) over the Nernst value of 59 mV per pH at room temperature when used as a biosensor. The transistors, which are fabricated from monolayer films of MoS2, use a room temperature ionic liquid (RTIL) in place of a conventional oxide gate dielectric and exhibit very low intrinsic noise resulting in a pH resolution of 92 × 10-6 at 10 Hz. This high device performance, which is a function of the structure of our device, is achieved by remotely connecting the gate to a pH sensing element allowing the FETs to be reused. Because pH measurements are fundamentally important in biotechnology, the increased resolution demonstrated here will benefit numerous applications ranging from pharmaceutical manufacturing to clinical diagnostics. As an example, we experimentally quantified the function of the kinase Cdk5, an enzyme implicated in Alzheimer's disease, at concentrations that are 5-fold lower than physiological values, and with sufficient time-resolution to allow the estimation of both steady-state and kinetic parameters in a single experiment. The high sensitivity, increased resolution, and fast turnaround time of the measurements will allow the development of early diagnostic tools and novel therapeutics to detect and treat neurological conditions years before currently possible.

Experimental validation of design and performance parameters of radioactive particle tracking (RPT) experimentation.

In recent years, radioactive particle tracking (RPT) has emerged as a powerful noninvasive technique for characterization and visualization of flow in opaque multiphase flow reactors. This technique has been applied to a variety of multiphase flow reactors largely based on the theoretical framework for optimal design and performance parameters. No systematic evaluation and validation of the design and performance parameters of the RPT technique has been reported in the literature thus far. Consequently, the theoretical framework for the design of RPT experiments has had limited scalability and application to a wide variety of flow systems. Thus far, design of a "good" RPT experiment continues to be an art, no matter how much the richness of flow of information that the experimental method brings. The present work reports systematic experimental evaluation of design parameters for an optimal RPT experiment and validation of the theoretical results reported in literature. The experiments were performed in a carefully designed setup in which precise positioning of the tracer particle was made possible. The experiments assess the effect of various parameters on the performance of the RPT experiment, such as the choice of radioactive isotope, activity, gamma-ray energy, size of the detector, and relative positioning of detectors. Finally, a set of recommendations based on experimental work are provided to "optimally" perform the RPT experiment in any single or multiphase reactor.

Overexpression of the Cdk5 inhibitory peptide in motor neurons rescue of amyotrophic lateral sclerosis phenotype in a mouse model.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects motor nerve cells in the brain and the spinal cord. Etiological mechanisms underlying the disease remain poorly understood; recent studies suggest that deregulation of p25/Cyclin-dependent kinase 5 (Cdk5) activity leads to the hyperphosphorylation of Tau and neurofilament (NF) proteins in ALS transgenic mouse model (SOD1G37R). A Cdk5 involvement in motor neuron degeneration is supported by analysis of three SOD1G37R mouse lines exhibiting perikaryal inclusions of NF proteins and hyperphosphorylation of Tau. Here, we tested the hypothesis that inhibition of Cdk5/p25 hyperactivation in vivo is a neuroprotective factor during ALS pathogenesis by crossing the new transgenic mouse line that overexpresses Cdk5 inhibitory peptide (CIP) in motor neurons with the SOD1G37R, ALS mouse model (TriTg mouse line). The overexpression of CIP in the motor neurons significantly improves motor deficits, extends survival and delays pathology in brain and spinal cord of TriTg mice. In addition, overexpression of CIP in motor neurons significantly delays neuroinflammatory responses in TriTg mouse. Taken together, these data suggest that CIP may serve as a novel therapeutic agent for the treatment of neurodegenerative diseases.

Evaluation of hydraulic performance of an anaerobic pond using radiotracer technique.

A radiotracer investigation was carried out in a wastewater stabilization pond using radiotracer technique. Residence time distribution (RTD) of wastewater was measured using Iodine-131 as a radiotracer. The measured RTD was treated and mean residence time (MRT) was determined. The measured MRT was compared with the theoretical MRT and dead volume was estimated. It was found that about 93% of the geometric volume within the anaerobic pond was dead. The measured RTD was modeled using various RTD models and eventually a suitable model was identified to describe and visualize the flow pattern of wastewater within the pond. Based on the measured MRT, estimated dead volume and identified flow patterns, it was concluded that the hydraulic performance of the anaerobic pond was very poor and not suitable for its intended purpose.