Anillin and the microtubule bundler PRC1 maintain myosin in the contractile ring to ensure completion of cytokinesis.

Cytokinesis is the last step of cell division, when one cell physically divides into two cells. Cytokinesis is driven by an equatorial contractile ring and signals from antiparallel microtubule bundles (the central spindle) that form between the two masses of segregating chromosomes. Bundling of central spindle microtubules is essential for cytokinesis in cultured cells. Using a temperature-sensitive mutant of SPD-1, the homolog of the microtubule bundler PRC1, we demonstrate that SPD-1 is required for robust cytokinesis in the Caenorhabditis elegans early embryo. SPD-1 inhibition results in broadening of the contractile ring, creating an elongated intercellular bridge between sister cells at the last stages of ring constriction that fails to seal. Moreover, depleting anillin/ANI-1 in SPD-1-inhibited cells results in myosin loss from the contractile ring during the second half of furrow ingression, which in turn results in furrow regression and cytokinesis failure. Our results thus reveal a mechanism involving the joint action of anillin and PRC1, which operates during the later stages of furrow ingression to ensure continued functioning of the contractile ring until cytokinesis is complete.

Ultraviolet Photodissociation and Activated Electron Photodetachment Mass Spectrometry for Top-Down Sequencing of Modified Oligoribonucleotides.

With the increased development of new RNA-based therapeutics, the need for robust analytical methods for confirming sequences and mapping modifications has accelerated. Characterizing modified ribonucleic acids using mass spectrometry is challenging because diagnostic fragmentation may be suppressed for modified nucleotides, thus hampering complete sequence coverage and the confident localization of modifications. Ultraviolet photodissociation (UVPD) has shown great potential for the characterization of nucleic acids due to extensive backbone fragmentation. Activated electron photodetachment dissociation (a-EPD) has also been used as an alternative to capitalize on the dominant charge-reduction pathway prevalent in UVPD, facilitate dissociation, and produce high abundances of fragment ions. Here, we compare higher-energy collisional activation (HCD), UVPD using 193 and 213 nm photons, and a-EPD for the top-down sequencing of modified nucleic acids, including methylated, phosphorothioate, and locked nucleic acid-modified DNA. The presence of these modifications alters the fragmentation pathways observed upon UVPD and a-EPD, and extensive backbone cleavage is observed that results in the production of fragment ions that retain the modifications and allow them to be pinpointed. LNA and 2'-O-methoxy phosphorothioate modifications caused a significant suppression of fragmentation for UVPD but not for a-EPD, whereas phosphorothioate bonds did not cause any significant suppression for either method. The incorporation of 2'-O-methyl modifications suppressed fragmentation of the antisense strand of patisiran, which resulted in some gaps in sequence coverage. However, UVPD provided the highest sequence coverage when compared to a-EPD.

Influence of Primary Structure on Fragmentation of Native-Like Proteins by Ultraviolet Photodissociation.

Analysis of native-like protein structures in the gas phase via native mass spectrometry and auxiliary techniques has become a powerful tool for structural biology applications. In combination with ultraviolet photodissociation (UVPD), native top-down mass spectrometry informs backbone flexibility, topology, hydrogen bonding networks, and conformational changes in protein structure. Although it is known that the primary structure affects dissociation of peptides and proteins in the gas phase, its effect on the types and locations of backbone cleavages promoted by UVPD and concomitant influence on structural characterization of native-like proteins is not well understood. Here, trends in the fragmentation of native-like proteins were evaluated by tracking the propensity of 10 fragment types (a, a+1, b, c, x, x+1, y, y-1, Y, and z) in relation to primary structure in a native-top down UVPD data set encompassing >9600 fragment ions. Differing fragmentation trends are reported for the production of distinct fragment types, attributed to a combination of both direct dissociation pathways from excited electronic states and those surmised to involve intramolecular vibrational energy redistribution after internal conversion. The latter pathways were systematically evaluated to evince the role of proton mobility in the generation of "CID-like" fragments through UVPD, providing pertinent insight into the characterization of native-like proteins. Fragmentation trends presented here are envisioned to enhance analysis of the protein higher-order structure or augment scoring algorithms in the high-throughput analysis of intact proteins.

Structural Characterization of Carbonic Anhydrase-Arylsulfonamide Complexes Using Ultraviolet Photodissociation Mass Spectrometry.

Numerous mass spectrometry-based strategies ranging from hydrogen-deuterium exchange to ion mobility to native mass spectrometry have been developed to advance biophysical and structural characterization of protein conformations and determination of protein-ligand interactions. In this study, we focus on the use of ultraviolet photodissociation (UVPD) to examine the structure of human carbonic anhydrase II (hCAII) and its interactions with arylsulfonamide inhibitors. Carbonic anhydrase, which catalyzes the conversion of carbon dioxide to bicarbonate, has been the target of countless thermodynamic and kinetic studies owing to its well-characterized active site, binding cavity, and mechanism of inhibition by hundreds of ligands. Here, we showcase the application of UVPD for evaluating structural changes of hCAII upon ligand binding on the basis of variations in fragmentation of hCAII versus hCAII-arylsulfonamide complexes, particularly focusing on the hydrophobic pocket. To extend the coverage in the midregion of the protein sequence, a supercharging agent was added to the solutions to increase the charge states of the complexes. The three arylsulfonamides examined in this study largely shift the fragmentation patterns in similar ways, despite their differences in binding affinities.

Recent developments in the characterization of nucleic acids by liquid chromatography, capillary electrophoresis, ion mobility, and mass spectrometry (2010-2020).

The development of new strategies for the analysis of nucleic acids has gained momentum due to the increased interest in using these biomolecules as drugs or drug targets. The application of new mass spectrometry ion activation techniques and the optimization of separation methods including liquid chromatography, capillary electrophoresis, and ion mobility have allowed more detailed characterization of nucleic acids and oligonucleotide therapeutics including confirmation of sequence, localization of modifications and interaction sites, and structural analysis as well as identification of failed sequences and degradation products. This review will cover tandem mass spectrometry methods as well as the recent developments in liquid chromatography, capillary electrophoresis, and ion mobility coupled to mass spectrometry for the analysis of nucleic acids and oligonucleotides.

Structural basis for assembly of non-canonical small subunits into type I-C Cascade.

Bacteria and archaea employ CRISPR (clustered, regularly, interspaced, short palindromic repeats)-Cas (CRISPR-associated) systems as a type of adaptive immunity to target and degrade foreign nucleic acids. While a myriad of CRISPR-Cas systems have been identified to date, type I-C is one of the most commonly found subtypes in nature. Interestingly, the type I-C system employs a minimal Cascade effector complex, which encodes only three unique subunits in its operon. Here, we present a 3.1 Å resolution cryo-EM structure of the Desulfovibrio vulgaris type I-C Cascade, revealing the molecular mechanisms that underlie RNA-directed complex assembly. We demonstrate how this minimal Cascade utilizes previously overlooked, non-canonical small subunits to stabilize R-loop formation. Furthermore, we describe putative PAM and Cas3 binding sites. These findings provide the structural basis for harnessing the type I-C Cascade as a genome-engineering tool.

A review of the analytical methods used for beer ingredient and finished product analysis and quality control.

Beer is an incredibly complex beverage containing more than 3000 different compounds, including carbohydrates, proteins, ions, microbes, organic acids, and polyphenols, among others. Beer becomes even more complex during storage, for over time it may undergo chemical changes that negatively affect the flavor, aroma, and appearance. Thus, it can be expected that maintaining the quality of beer throughout its lifetime is a difficult task. Since it is such a popular drink throughout the world, being familiar with proper analytical techniques for beer evaluation is useful for researchers and brewers. These techniques include, but are not limited to, gas chromatography, liquid chromatography, matrix assisted laser desorption/ionization, capillary electrophoresis, mass spectrometry, ultraviolet-visible spectroscopy, and flame ionization detection. This review aims to summarize the various ingredients and components of beer, discuss how they affect the finished product, and present some of the analytical methods used for quality control and understanding the formation of chemicals in beer during the brewing process.

Large-volume injection gas chromatography-vacuum ultraviolet spectroscopy for the qualitative and quantitative analysis of fatty acids in blood plasma.

Qualitative and quantitative determination of fatty acids in plasma is of extreme importance as these are indicators of metabolic diseases. In this work, a sensitive and rugged method for detecting and quantifying fatty acids (as fatty acid methyl ester derivatives, FAMEs) in blood plasma was developed. The use of large-volume injection (LVI) gas chromatography-vacuum ultraviolet spectroscopy (GC-VUV) for analysis of fatty acids in blood plasma allowed the injection of higher sample volumes to accommodate sufficient analyte on-column for necessary detection ranges with a run time of 45 min. Calibration curves exhibited consistent linearity and reproducibility and were used along with internal standards for the quantification of 11 saturated and 21 unsaturated fatty acids. Intra-day and inter-day (n = 6) CVs had an average of 5 and 6%, respectively, and recoveries an average of 105%. The concentrations of EPA, DHA, and AA, as well as the omega-3 index and omega-6/omega-3 ratio, were calculated and compared with clinically actionable measurement ranges. Due to the use of LVI, the more volatile analyte (C8:0) was lost and therefore impossible to quantify. The volatility cutoff was determined to be the C10:0 analyte with a molecular weight of 186.295 g/mol.

Pull-Down of Metalloproteins in Their Native States by Using Desthiobiotin-Based Probes.

One-third of all proteins are estimated to require metals for structural stability and/or catalytic activity. Desthiobiotin probes containing metal binding groups can be used to capture metalloproteins with exposed active-site metals under mild conditions so as to prevent changes in metallation state. The proof-of-concept was demonstrated with carbonic anhydrase (CA), an open active site, Zn2+ -containing protein. CA was targeted by using sulfonamide derivatives. Linkers of various lengths and structures were screened to determine the optimal structure for capture of the native protein. The optimized probes could selectively pull down CA from red blood cell lysate and other protein mixtures. Pull-down of differently metallated CAs was also investigated.

Analysis of bacteria stress responses to contaminants derived from shale energy extraction.

In order to survive environmental changes, bacteria have stress responses, which protect them from adverse and variable conditions. Contamination can be a source of stress and bacterial responses can serve as an indicator of environmental abnormality. In this work, the biochemical effects of toxic compounds that stem from hydraulic fracturing were measured on the whole cell-derived fatty acid and protein compositions of Escherichia coli, Klebsiella oxytoca, Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas stutzeri, Aeromonas hydrophila, Bacillus cereus, and Bacillus subtilis. These microorganisms were exposed to elevated levels of benzene, ethanol, propanol, toluene, and salt. These were chosen to represent significant subsurface contamination or a surface spill. The fatty acid and protein profiles for the bacteria were analyzed using gas chromatography - vacuum ultraviolet spectroscopy and matrix-assisted laser desorption ionization time-of-flight mass spectrometry, respectively. Overall, different fatty acid and protein profiles were observed when the microorganisms were grown in the presence of the toxic compounds. The cells exhibited an increase in the saturated/unsaturated ratio and displayed the presence of branched and cyclopropane fatty acids when in the presence of common fracture fluid constituents to decrease membrane permeability which was confirmed by the analysis of produced water. This approach provides a potentially useful tool for environmental diagnosis, since proteins and fatty acids can act as a harbinger of ecological health.

Low- and high-threshold primary afferent inputs to spinal lamina III antenna-type neurons.

The dorsal horn of the spinal cord (laminae I-VI) processes diverse modalities of nociceptive and nonnociceptive sensory information. Antenna-type neurons with cell bodies located in lamina III and large dendritic trees extending from the superficial lamina I to deep lamina IV are best shaped for the integration of a wide variety of inputs arising from primary afferent fibers and intrinsic spinal circuitries. Although the somatodendritic morphology, the hallmark of antenna neurons, has been well studied, little is still known about the axon structure and basic physiological properties of these cells. Here, we did whole-cell recordings in a rat (P9-P12) spinal cord preparation with attached dorsal roots to examine the axon course, intrinsic firing properties, and primary afferent inputs of antenna cells. Nine antenna cells were identified from a large sample of biocytin-filled lamina III neurons (n = 46). Axon of antenna cells showed intensive branching in laminae III-IV and, in half of the cases, issued dorsally directed collaterals reaching lamina I. Antenna cells exhibited tonic and rhythmic firing patterns; single spikes were followed by hyperpolarization or depolarization. The neurons received monosynaptic inputs from the low-threshold Aß afferents, Aδ afferents, as well as from the high-threshold Aδ, and C afferents. When selectively activated, C-fiber-driven monosynaptic and polysynaptic excitatory postsynaptic potentials were sufficiently strong to evoke firing in the neurons. Thus, lamina III antenna neurons integrate low-threshold and nociceptive high-threshold primary afferent inputs and can function as wide dynamic range neurons able to directly connect deep dorsal horn with the major nociceptive projection area lamina I.

NaCl-saturated brines are thermodynamically moderate, rather than extreme, microbial habitats.

NaCl-saturated brines such as saltern crystalliser ponds, inland salt lakes, deep-sea brines and liquids-of-deliquescence on halite are commonly regarded as a paradigm for the limit of life on Earth. There are, however, other habitats that are thermodynamically more extreme. Typically, NaCl-saturated environments contain all domains of life and perform complete biogeochemical cycling. Despite their reduced water activity, ∼0.755 at 5 M NaCl, some halophiles belonging to the Archaea and Bacteria exhibit optimum growth/metabolism in these brines. Furthermore, the recognised water-activity limit for microbial function, ∼0.585 for some strains of fungi, lies far below 0.755. Other biophysical constraints on the microbial biosphere (temperatures of >121°C; pH > 12; and high chaotropicity; e.g. ethanol at >18.9% w/v (24% v/v) and MgCl2 at >3.03 M) can prevent any cellular metabolism or ecosystem function. By contrast, NaCl-saturated environments contain biomass-dense, metabolically diverse, highly active and complex microbial ecosystems; and this underscores their moderate character. Here, we survey the evidence that NaCl-saturated brines are biologically permissive, fertile habitats that are thermodynamically mid-range rather than extreme. Indeed, were NaCl sufficiently soluble, some halophiles might grow at concentrations of up to 8 M. It may be that the finite solubility of NaCl has stabilised the genetic composition of halophile populations and limited the action of natural selection in driving halophile evolution towards greater xerophilicity. Further implications are considered for the origin(s) of life and other aspects of astrobiology.

Treatment modalities for the reuse of produced waste from oil and gas development.

Unconventional oil and gas development is achieved through a series of sub-processes, which utilize large amounts of water, proppant, and chemical additives to retrieve sequestered hydrocarbons from low permeability petroliferous strata. As a result, a large amount of wastewater is produced, which is traditionally disposed of via subsurface injection into non-productive stratum throughout the country. However, this method of waste management has been linked to the induction of seismic events in a number of regions across North America, calling into question the environmental stewardship and sustainability of subsurface waste disposal. Advancements in water treatment technologies have improved the efficacy and financial viability of produced water recycling for beneficial reuse in the oil and gas sector. This review will cover the various treatment options that are currently being utilized in shale energy basins to remove organic, inorganic, and biological constituents, as well as some emerging technologies that are designed to remove pertinent contaminants that would otherwise preclude the reuse of produced water for production well stimulation.

Characterizing variable biogeochemical changes during the treatment of produced oilfield waste.

At the forefront of the discussions about climate change and energy independence has been the process of hydraulic fracturing, which utilizes large amounts of water, proppants, and chemical additives to stimulate sequestered hydrocarbons from impermeable subsurface strata. This process also produces large amounts of heterogeneous flowback and formation waters, the subsurface disposal of which has most recently been linked to the induction of anthropogenic earthquakes. As such, the management of these waste streams has provided a newfound impetus to explore recycling alternatives to reduce the reliance on subsurface disposal and fresh water resources. However, the biogeochemical characteristics of produced oilfield waste render its recycling and reutilization for production well stimulation a substantial challenge. Here we present a comprehensive analysis of produced waste from the Eagle Ford shale region before, during, and after treatment through adjustable separation, flocculation, and disinfection technologies. The collection of bulk measurements revealed significant reductions in suspended and dissolved constituents that could otherwise preclude untreated produced water from being utilized for production well stimulation. Additionally, a significant step-wise reduction in pertinent scaling and well-fouling elements was observed, in conjunction with notable fluctuations in the microbiomes of highly variable produced waters. Collectively, these data provide insight into the efficacies of available water treatment modalities within the shale energy sector, which is currently challenged with improving the environmental stewardship of produced water management.

Analysis of bacterial FAMEs using gas chromatography - vacuum ultraviolet spectroscopy for the identification and discrimination of bacteria.

The identification of microorganisms is very important in different fields and alternative methods are necessary for a rapid and simple identification. The use of fatty acids for bacterial identification is gaining attention as phenotypic characteristics are reflective of the genotype and are more easily analyzed. In this work, gas chromatography-vacuum ultraviolet spectroscopy (GC-VUV) was used to determine bacteria fatty acid methyl esters (FAMEs), to identify and discriminate different environmental bacteria based on their fatty acid profile. Microorganisms were grown in agar and their fatty acids extracted, saponified, and esterified before analysis. Unique FAME profiles were obtained for each microorganism mainly composed of branched, cyclopropane, hydroxy, saturated, and unsaturated fatty acid methyl esters. S. maltophilia showed a higher diversity of fatty acids while Bacillus species showed higher complexity in terms of branched-chain FAMEs, with several iso and anteiso forms. 12 different bacteria genera and 15 species were successfully differentiated based on their fatty acid profiles after performing PCA and cluster analysis. Some difficult to differentiate species, such as Bacillus sp., which are genetically very similar, were differentiated with the developed method.

Exploring the links between groundwater quality and bacterial communities near oil and gas extraction activities.

Bacterial communities in groundwater are very important as they maintain a balanced biogeochemical environment. When subjected to stressful environments, for example, due to anthropogenic contamination, bacterial communities and their dynamics change. Studying the responses of the groundwater microbiome in the face of environmental changes can add to our growing knowledge of microbial ecology, which can be utilized for the development of novel bioremediation strategies. High-throughput and simpler techniques that allow the real-time study of different microbiomes and their dynamics are necessary, especially when examining larger data sets. Matrix-assisted laser desorption-ionization (MALDI) time-of-flight mass spectrometry (TOF-MS) is a workhorse for the high-throughput identification of bacteria. In this work, groundwater samples were collected from a rural area in southern Texas, where agricultural activities and unconventional oil and gas development are the most prevalent anthropogenic activities. Bacterial communities were assessed using MALDI-TOF MS, with bacterial diversity and abundance being analyzed with the contexts of numerous organic and inorganic groundwater constituents. Mainly denitrifying and heterotrophic bacteria from the Phylum Proteobacteria were isolated. These microorganisms are able to either transform nitrate into gaseous forms of nitrogen or degrade organic compounds such as hydrocarbons. Overall, the bacterial communities varied significantly with respect to the compositional differences that were observed from the collected groundwater samples. Collectively, these data provide a baseline measurement of bacterial diversity in groundwater located near anthropogenic surface and subsurface activities.

Characterization of bacterial diversity in contaminated groundwater using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Groundwater is a major source for drinking water in the United States, and therefore, its quality and quantity is of extreme importance. One major concern that has emerged is the possible contamination of groundwater due to the unconventional oil and gas extraction activities. As such, the impacts of exogenous contaminants on microbial ecology is an area to be explored to understand what are the chemical and physical conditions that allow the proliferation of pathogenic bacteria and to find alternatives for water treatment by identifying organic-degrading bacteria. In this work, we assess the interplay between groundwater quality and the microbiome in contaminated groundwaters rich in hydrocarbon gases, volatile organic and inorganic compounds, and various metals. Opportunistic pathogenic bacteria, such as Aeromonas hydrophila, Bacillus cereus, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia, were identified, increasing the risk for consumption of and exposure to these contaminated groundwaters. Additionally, antimicrobial tests revealed that many of the identified bacteria were resistant to different antibiotics. The MALDI-TOF MS results were successfully confirmed with 16S rRNA gene sequencing, proving the accuracy of this high-throughput method. Collectively, these data provide a seminal understanding of the microbial populations in contaminated groundwater overlying anthropogenic activities like unconventional oil and gas development.

MALDI-TOF MS for the Identification of Cultivable Organic-Degrading Bacteria in Contaminated Groundwater near Unconventional Natural Gas Extraction Sites.

Groundwater quality and quantity is of extreme importance as it is a source of drinking water in the United States. One major concern has emerged due to the possible contamination of groundwater from unconventional oil and natural gas extraction activities. Recent studies have been performed to understand if these activities are causing groundwater contamination, particularly with respect to exogenous hydrocarbons and volatile organic compounds. The impact of contaminants on microbial ecology is an area to be explored as alternatives for water treatment are necessary. In this work, we identified cultivable organic-degrading bacteria in groundwater in close proximity to unconventional natural gas extraction. Pseudomonas stutzeri and Acinetobacter haemolyticus were identified using matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF MS), which proved to be a simple, fast, and reliable method. Additionally, the potential use of the identified bacteria in water and/or wastewater bioremediation was studied by determining the ability of these microorganisms to degrade toluene and chloroform. In fact, these bacteria can be potentially applied for in situ bioremediation of contaminated water and wastewater treatment, as they were able to degrade both compounds.