Uncoupling the TFIIH Core and Kinase Modules Leads To Unregulated RNA Polymerase II CTD Serine 5 Phosphorylation.

TFIIH is an essential transcription initiation factor for RNA polymerase II (RNApII). This multi-subunit complex comprises two modules that are physically linked by the subunit Tfb3 (MAT1 in metazoans). The TFIIH Core Module, with two DNA-dependent ATPases and several additional subunits, promotes DNA unwinding. The TFIIH Kinase Module phosphorylates Serine 5 of the C-terminal domain (CTD) of RNApII subunit Rpb1, a modification that coordinates exchange of initiation and early elongation factors. While it is not obvious why these two disparate activities are bundled into one factor, the connection may provide temporal coordination during early initiation. Here we show that Tfb3 can be split into two parts to uncouple the TFIIH modules. The resulting cells grow slower than normal, but are viable. Chromatin immunoprecipitation of the split TFIIH shows that the Core Module, but not the Kinase, is properly recruited to promoters. Instead of the normal promoter-proximal peak, high CTD Serine 5 phosphorylation is seen throughout transcribed regions. Therefore, coupling the TFIIH modules is necessary to localize and limit CTD kinase activity to early stages of transcription. These results are consistent with the idea that the two TFIIH modules began as independent functional entities that became connected by Tfb3 during early eukaryotic evolution.

Scalable and green formation of graphitic nanolayers produces highly conductive pyrolyzed paper toward sensitive electrochemical sensors.

While pyrolyzed paper (PP) is a green and abundant material that can provide functionalized electrodes with wide detection windows for a plethora of targets, it poses long-standing challenges against sensing assays such as poor electrical conductivity, with resistivities generally higher than 200.0 mΩ cm (e.g., gold and silver show resistivities 1000-fold lower, ∼0.2 mΩ cm). In this regard, the fundamental hypothesis that drives this work is whether a scalable, cost-effective, and eco-friendly strategy is capable of significantly reducing the resistivity of PP electrodes toward the development of sensitive electrochemical sensors, whether faradaic or capacitive. We address this hypothesis by simply annealing PP under an isopropanol atmosphere for 1 h, reaching resistivities as low as 7 mΩ cm. Specifically, the annealing of PP at 800 or 1000 °C under isopropanol vapor leads to the formation of a highly graphitic nanolayer (∼15 nm) on the PP surface, boosting conductivity as the delocalization of π electrons stemming from carbon sp2 is favored. The reduction of carbonyl groups and the deposition of dehydrated isopropanol during the annealing process are hypothesized herein as the dominant PP graphitization mechanisms. Electrochemical analyses demonstrated the capability of the annealed PP to increase the charge-transfer kinetics, with the optimum heterogeneous standard rate constant being roughly 3.6 × 10-3 cm s-1. This value is larger than the constants reported for other carbon electrodes and indium tin oxide. Furthermore, freestanding fingers of the annealed PP were prototyped using a knife plotter to fabricate impedimetric on-leaf electrodes. These wearable sensors ensured the real-time and in situ monitoring of the loss of water content from soy leaves, outperforming non-annealed electrodes in terms of reproducibility and sensitivity. Such an application is of pivotal importance for precision agriculture and development of agricultural inputs. This work addresses the foundations for the achievement of conductive PP in a scalable, low-cost, simple, and eco-friendly way, i.e. without producing any liquid chemical waste, providing new opportunities to translate PP-based sensitive electrochemical devices into practical use.

Machine learning toward high-performance electrochemical sensors.

The so-coined fourth paradigm in science has reached the sensing area, with the use of machine learning (ML) toward data-driven improvements in sensitivity, reproducibility, and accuracy, along with the determination of multiple targets from a single measurement using multi-output regression models. Particularly, the use of supervised ML models trained on large data sets produced by electrical and electrochemical bio/sensors has emerged as an impacting trend in the literature by allowing accurate analyses even in the presence of usual issues such as electrode fouling, poor signal-to-noise ratio, chemical interferences, and matrix effects. In this trend article, apart from an outlook for the coming years, we present examples from the literature that demonstrate how helpful ML algorithms can be for dispensing the adoption of experimental methods to address the aforesaid interfering issues, ultimately contributing to translate testing technologies into on-site, practical, and daily applications.

Real-Time and In Situ Monitoring of the Synthesis of Silica Nanoparticles.

The real-time and in situ monitoring of the synthesis of nanomaterials (NMs) remains a challenging task, which is of pivotal importance by assisting fundamental studies (e.g., synthesis kinetics and colloidal phenomena) and providing optimized quality control. In fact, the lack of reproducibility in the synthesis of NMs is a bottleneck against the translation of nanotechnologies into the market toward daily practice. Here, we address an impedimetric millifluidic sensor with data processing by machine learning (ML) as a sensing platform to monitor silica nanoparticles (SiO2NPs) over a 24 h synthesis from a single measurement. The SiO2NPs were selected as a model NM because of their extensive applications. Impressively, simple ML-fitted descriptors were capable of overcoming interferences derived from SiO2NP adsorption over the signals of polarizable Au interdigitate electrodes to assure the determination of the size and concentration of nanoparticles over synthesis while meeting the trade-off between accuracy and speed/simplicity of computation. The root-mean-square errors were calculated as ∼2.0 nm (size) and 2.6 × 1010 nanoparticles mL-1 (concentration). Further, the robustness of the ML size descriptor was successfully challenged in data obtained along independent syntheses using different devices, with the global average accuracy being 103.7 ± 1.9%. Our work advances the developments required to transform a closed flow system basically encompassing the reactional flask and an impedimetric sensor into a scalable and user-friendly platform to assess the in situ synthesis of SiO2NPs. Since the sensor presents a universal response principle, the method is expected to enable the monitoring of other NMs. Such a platform may help to pave the way for translating "sense-act" systems into practice use in nanotechnology.

The O-Ag Antibody Response to Francisella Is Distinct in Rodents and Higher Animals and Can Serve as a Correlate of Protection.

Identifying correlates of protection (COPs) for vaccines against lethal human (Hu) pathogens, such as Francisella tularensis (Ft), is problematic, as clinical trials are currently untenable and the relevance of various animal models can be controversial. Previously, Hu trials with the live vaccine strain (LVS) demonstrated ~80% vaccine efficacy against low dose (~50 CFU) challenge; however, protection deteriorated with higher challenge doses (~2000 CFU of SchuS4) and no COPs were established. Here, we describe our efforts to develop clinically relevant, humoral COPs applicable to high-dose, aerosol challenge with S4. First, our serosurvey of LVS-vaccinated Hu and animals revealed that rabbits (Rbs), but not rodents, recapitulate the Hu O-Ag dependent Ab response to Ft. Next, we assayed Rbs immunized with distinct S4-based vaccine candidates (S4ΔclpB, S4ΔguaBA, and S4ΔaroD) and found that, across multiple vaccines, the %O-Ag dep Ab trended with vaccine efficacy. Among S4ΔguaBA-vaccinated Rbs, the %O-Ag dep Ab in pre-challenge plasma was significantly higher in survivors than in non-survivors; a cut-off of >70% O-Ag dep Ab predicted survival with high sensitivity and specificity. Finally, we found this COP in 80% of LVS-vaccinated Hu plasma samples as expected for a vaccine with 80% Hu efficacy. Collectively, the %O-Ag dep Ab response is a bona fide COP for S4ΔguaBA-vaccinated Rb and holds significant promise for guiding vaccine trials with higher animals.

Bifunctional Metal Meshes Acting as a Semipermeable Membrane and Electrode for Sensitive Electrochemical Determination of Volatile Compounds.

The monitoring of toxic inorganic gases and volatile organic compounds has brought the development of field-deployable, sensitive, and scalable sensors into focus. Here, we attempted to meet these requirements by using concurrently microhole-structured meshes as (i) a membrane for the gas diffusion extraction of an analyte from a donor sample and (ii) an electrode for the sensitive electrochemical determination of this target with the receptor electrolyte at rest. We used two types of meshes with complementary benefits, i.e., Ni mesh fabricated by robust, scalable, and well-established methods for manufacturing specific designs and stainless steel wire mesh (SSWM), which is commercially available at a low cost. The diffusion of gas (from a donor) was conducted in headspace mode, thus minimizing issues related to mesh fouling. When compared with the conventional polytetrafluoroethylene (PTFE) membrane, both the meshes (40 µm hole diameter) led to a higher amount of vapor collected into the electrolyte for subsequent detection. This inedited fashion produced a kind of reverse diffusion of the analyte dissolved into the electrolyte (receptor), i.e., from the electrode to bulk, which further enabled highly sensitive analyses. Using Ni mesh coated with Ni(OH)2 nanoparticles, the limit of detection reached for ethanol was 24-fold lower than the data attained by a platform with a PTFE membrane and placement of the electrode into electrolyte bulk. This system was applied in the determination of ethanol in complex samples related to the production of ethanol biofuel. It is noteworthy that a simple equation fitted by machine learning was able to provide accurate assays (accuracies from 97 to 102%) by overcoming matrix effect-related interferences on detection performance. Furthermore, preliminary measurements demonstrated the successful coating of the meshes with gold films as an alternative raw electrode material and the monitoring of HCl utilizing Au-coated SSWMs. These strategies extend the applicability of the platform that may help to develop valuable volatile sensing solutions.

Genetic Analysis of DinG Family Helicase YoaA and Its Interaction with Replication Clamp Loader Protein HolC in Escherichia coli.

The XP-D/DinG family of DNA helicases contributes to genomic stability in all three domains of life. Here, we investigate the role of one of these proteins, YoaA, of Escherichia coli. In E. coli, YoaA aids in tolerance to the nucleoside azidothymidine (AZT), a DNA replication inhibitor, and physically interacts with a subunit of the DNA polymerase III holoenzyme, HolC. We map the residues of YoaA required for HolC interaction to its C terminus by yeast two-hybrid analysis. We propose that this interaction competes with HolC's interaction with HolD and the rest of the replisome; YoaA indeed inhibits growth when overexpressed, dependent on this interaction region. By gene fusions, we show that YoaA is repressed by LexA and induced in response to DNA damage as part of the SOS response. Induction of YoaA by AZT is biphasic, with an immediate response after treatment and a slower response that peaks in the late log phase of growth. This growth-phase-dependent induction by AZT is not blocked by lexA3 (Ind-), which normally negates its self-cleavage, implying another means to induce the DNA damage response that responds to the nutritional state of the cell. We propose that YoaA helicase activity increases access to the 3' nascent strand during replication; consistent with this, YoaA appears to aid in the removal of potential A-to-T transversion mutations in ndk mutants, which are prone to nucleotide misincorporation. We provide evidence that YoaA and its paralog DinG may also initiate template switching that leads to deletions between tandem repeats in DNA. IMPORTANCE Maintaining genomic stability is crucial for all living organisms. Replication of DNA frequently encounters barriers that must be removed to complete genome duplication. Balancing DNA synthesis with its repair is critical and not entirely understood at a mechanistic level. The YoaA protein, studied here, is required for certain types of DNA repair and interacts in an alternative manner with proteins that catalyze DNA replication. YoaA is part of the well-studied LexA-regulated response to DNA damage, the SOS response. We describe an unusual feature of its regulation that promotes induction after DNA damage as the culture begins to experience starvation. Replication fork repair integrates both DNA damage and nutritional signals. We also show that YoaA affects genomic stability.

Gravity-assisted distillation on a chip: Fabrication, characterization, and applications.

Distillation is widely used in industrial processes and laboratories for sample pre-treatment. The conventional apparatus of flash distillation is composed of heating source, distilling flask, condenser, and receiving flask. As disadvantages, this method shows manual and laborious analyses with high consumption of chemicals. In this paper, all these limitations were addressed by developing a fully integrated microscale distiller in agreement with the apparatus of conventional flash distillation. The main challenge facing the distillation miniaturization is the phase separation since surface forces take over from the gravity in microscale channels. Otherwise, our chip had ability to perform gravity-assisted distillations because of the somewhat large dimensions of the distillation chamber (roughly 900 µL) that was obtained by 3D-printing. The functional distillation units were integrated into a single device composed of polydimethylsiloxane (PDMS). Its fabrication was cost-effective and simple by avoiding the use of cleanroom and bonding step. In addition to user-friendly analysis and low consumption of chemicals, the method requires cost-effective instrumentation, namely, voltage supply and analytical balance. Furthermore, the so called distillation-on-a-chip (DOC) eliminates the use of membranes and electrodes (usually employed in microfluidic desalinations reported in the literature), thus avoiding drawbacks such as liquid leakage, membrane fouling, and electrode passivation. The DOC promoted desalinations at harsh salinity (NaCl 600.0 mmol L-1) with high throughput and salt removal efficiency (roughly 99%). Besides, the method was used for determination of ethanol in alcoholic beverages to show the potential of the approach toward quantitative purposes.

Renewable Solid Electrodes in Microfluidics: Recovering the Electrochemical Activity without Treating the Surface.

The contamination, passivation, or fouling of the detection electrodes is a serious problem undermining the analytical performance of electroanalytical devices. The methods to regenerate the electrochemical activity of the solid electrodes involve mechanical, physical, or chemical surface treatments that usually add operational time, complexity, chemicals, and further instrumental requirements to the analysis. In this paper, we describe for the first time a reproducible method for renewing solid electrodes whenever their morphology or composition are nonspecifically changed without any surface treatment. These renewable electrodes are the closest analogue to the mercury drop electrodes. Our approach was applied in microfluidics, where the downsides related to nonspecific modifications of the electrode are more critical. The renewal consisted in manually sliding metal-coated microwires across a channel with the sample. For this purpose, the chip was composed of a single piece of polydimethylsiloxane (PDMS) with three parallel channels interconnected to one perpendicular and top channel. The microwires were inserted in each one of the parallel channels acting as working, counter, and pseudoreference electrodes for voltammetry. This assembly allowed the renewal of all the three electrodes by simply pulling the microwires. The absence of any interfaces in the chips and the elastomeric nature of the PDMS allowed us to pull the microwires without the occurrence of leakages for the electrode channels even at harsh flow rates of up to 40.0 mL min-1. We expect this paper can assist the researchers to develop new microfluidic platforms that eliminate any steps of electrode cleaning, representing a powerful alternative for precise and robust analyses to real samples.

Turbulence in microfluidics: Cleanroom-free, fast, solventless, and bondless fabrication and application in high throughput liquid-liquid extraction.

This paper addresses an important breakthrough in the deployment of ultra-high adhesion strength microfluidic technologies to provide turbulence at harsh flow rate conditions. This paper is only, to our knowledge, the second reporting on the generation of high flow rate-assisted turbulence in microchannels. This flow solves a crucial bottleneck in microfluidics: the generation of high throughput homogeneous mixings. We focused on the fabrication of bulky polydimethylsiloxane (PDMS) microchips (without any interfaces) rather than the laborious surface modifications that were employed in the first reporting about turbulence-assisted microfluidics. The fabrication is cleanroom-free, simple, low-cost, fast, solventless, and bondless requiring only a laboratory oven. More specifically, our method relies on the shaping of a nylon scaffold, cure of PDMS with embedded nylon, and removal of this scaffold. The scaffold was obtained by manually wrapping nylon threads. The withdrawing out of the scaffold was completed in few seconds using only a plier. Such microchannels endured flow rates of up to 60.0 mL min(-1) with a strikingly low elastic deformation. The importance in producing turbulence into microscale channels was successfully shown in liquid-liquid extractions. The great energy dissipation rate relative to the turbulence created high throughput and efficient extractions in microfluidics for the first time. The residence time was only 0.01 s at 25.0 mL min(-1) (total flow rate of the immiscible phases). In addition, the partition coefficient determined in a single run was similar to that obtained by the conventional batch shake-flask method that was realized in triplicate.

An integrated platform for gas-diffusion separation and electrochemical determination of ethanol on fermentation broths.

An integrated platform was developed for point-of-use determination of ethanol in sugar cane fermentation broths. Such analysis is important because ethanol reduces its fuel production efficiency by altering the alcoholic fermentation step when in excess. The custom-designed platform integrates gas diffusion separation with voltammetric detection in a single analysis module. The detector relied on a Ni(OH)2-modified electrode. It was stabilized by uniformly depositing cobalt and cadmium hydroxides as shown by XPS measurements. Such tests were in accordance with the hypothesis related to stabilization of the Ni(OH)2 structure by insertion of Co(2+) and Cd(2+) ions in this structure. The separation step, in turn, was based on a hydrophobic PTFE membrane, which separates the sample from receptor solution (electrolyte) where the electrodes were placed. Parameters of limit of detection and analytical sensitivity were estimated to be 0.2% v/v and 2.90 µA % (v/v)(-1), respectively. Samples of fermentation broth were analyzed by both standard addition method and direct interpolation in saline medium based-analytical curve. In this case, the saline solution exhibited ionic strength similar to those of the samples intended to surpass the tonometry colligative effect of the samples over analyte concentration data by attributing the reduction in quantity of diffused ethanol vapor majorly to the electrolyte. The approach of analytical curve provided rapid, simple and accurate analysis, thus contributing for deployment of point-of-use technologies. All of the results were accurate with respect to those obtained by FTIR method at 95% confidence level.

Optical paper-based sensor for ascorbic acid quantification using silver nanoparticles.

In this paper, we demonstrate for the first time the use of silver nanoparticles (AgNPs) for colorimetric ascorbic acid (AA) quantification in a paper-based sensor. This device is constituted by spot tests modified with AgNPs and silver ions bordered by a hydrophobic barrier which provides quantitative and fast analysis of AA. In addition, this device is employed as point-of-care monitoring using a unique drop of the sample. AgNPs paper-based sensor changed from light yellow to gray color after the addition of AA due to nanoparticle growth and clusters formation. The color intensities were altered as a function of AA concentration which were measured by either a scanner or a homemade portable transmittance colorimeter. Under the selected measurement conditions, results presented limit of detection which was comparable to analytical laboratory-based methodologies. In addition, the sensitivity of our sensor was comparable to the standard titration method when real samples were investigated.