Single-tube protocol for culture-independent spoligotyping of Mycobacterium tuberculosis based on MeltArray.

IMPORTANCE: Spacer oligonucleotide typing (spoligotyping), the first-line genotyping assay for Mycobacterium tuberculosis (MTB), plays a fundamental role in the investigation of its epidemiology and evolution. In this study, we established a single-tube spoligotyping assay using MeltArray, a highly multiplex polymerase chain reaction (PCR) approach that runs on a real-time PCR thermocycler. The MeltArray protocol included an internal positive control, gyrB, to indicate the abundance of MTB via the quantification cycle and 43 spacers to identify the spoligotype via melting curve analysis. The entire protocol was completed in a single step within 2.5 hours. The lowest detectable copy number for the tested strains was 20 copies/reaction and thus sufficient for analyzing both culture and sputum samples. We conclude that MeltArray-based spoligotyping could be used immediately in low- and middle-income countries with a high tuberculosis burden, given its easy access, improved throughput, and potential applicability to clinical samples.

The structure of the phage T4 DNA packaging motor suggests a mechanism dependent on electrostatic forces.

Viral genomes are packaged into "procapsids" by powerful molecular motors. We report the crystal structure of the DNA packaging motor protein, gene product 17 (gp17), in bacteriophage T4. The structure consists of an N-terminal ATPase domain, which provides energy for compacting DNA, and a C-terminal nuclease domain, which terminates packaging. We show that another function of the C-terminal domain is to translocate the genome into the procapsid. The two domains are in close contact in the crystal structure, representing a "tensed state." A cryo-electron microscopy reconstruction of the T4 procapsid complexed with gp17 shows that the packaging motor is a pentamer and that the domains within each monomer are spatially separated, representing a "relaxed state." These structures suggest a mechanism, supported by mutational and other data, in which electrostatic forces drive the DNA packaging by alternating between tensed and relaxed states. Similar mechanisms may occur in other molecular motors.

Polypropylene microplastics aging under natural conditions in winter and summer and its effects on the sorption and desorption of nonylphenol.

Plastics in the environment undergo various aging effects. Due to the changes in physical and chemical properties, the sorption behavior of aged microplastics (MPs) for pollutants differs from that of pristine MPs. In this paper, the most common disposable polypropylene (PP) rice box was used as the source of MPs to study the sorption and desorption behavior of nonylphenol (NP) on pristine and naturally aged PPs in summer and winter. The results show that summer-aged PP has more obvious property changes than winter-aged PP. The equilibrium sorption amount of NP on PP is summer-aged PP (477.08 µg/g) > winter-aged PP (407.14 µg/g) > pristine PP (389.29 µg/g). The sorption mechanism includes the partition effect, van der Waals forces, hydrogen bonds and hydrophobic interaction, among which chemical sorption (hydrogen bonding) dominates the sorption; moreover, partition also plays an important role in this process. Aged MPs' more robust sorption capacity is attributed to the larger specific surface area, stronger polarity and more oxygen-containing functional groups on the surface that are conducive to forming hydrogen bonds with NP. Desorption of NP in the simulated intestinal fluid is significant owning to intestinal micelles' presence: summer-aged PP (300.52 µg/g) > winter-aged PP (291.08 µg/g) > pristine PP (287.12 µg/g). Hence, aged PP presents a more vital ecological risk.

Removal of tetracyclines from aqueous solutions by electrocoagulation/pecan nutshell coupling processes: synergistic effect and mechanism.

The present work compared electrocoagulation (EC)/pecan shell (PS) coupling process with a simple electrocoagulation (EC) process for the removal of tetracyclines (TCs). The results indicated that the addition of appropriate PS could lead to the enhancement of the removal efficiency and decrease of operating time via synergistic influence, including conventional EC process, biomass materials adsorption, charge neutralization and coordination adsorption. The ideal condition for the coupling process was 2.5 mA/cm2 for current density and 3 cm for plate spacing. Based on the optimum condition, when the dosage of PS was 5 g/L, the initial concentration of tetracycline hydrochloride (TC), oxytetracycline hydrochloride (OTC) and chlortetracycline hydrochloride (CTC) was 250 mg/L, the removal rate of PS was 55.90%, 45.10% and 14.98% higher than those of EC process after 40 min treatment. In addition, compared to conventional EC process, the unit energy demand (UED) decreased by 49.62%, 53.2 4% and 26.35% and the unit electrode material demand (UEMD) decreased by 49.80%, 85.65% and 44.37%, respectively, which means more energy conservation and environmental protection.

Rubella virus capsid protein structure and its role in virus assembly and infection.

Rubella virus (RV) is a leading cause of birth defects due to infectious agents. When contracted during pregnancy, RV infection leads to severe damage in fetuses. Despite its medical importance, compared with the related alphaviruses, very little is known about the structure of RV. The RV capsid protein is an essential structural component of virions as well as a key factor in virus-host interactions. Here we describe three crystal structures of the structural domain of the RV capsid protein. The polypeptide fold of the RV capsid protomer has not been observed previously. Combining the atomic structure of the RV capsid protein with the cryoelectron tomograms of RV particles established a low-resolution structure of the virion. Mutational studies based on this structure confirmed the role of amino acid residues in the capsid that function in the assembly of infectious virions.

Structure of Sputnik, a virophage, at 3.5-Å resolution.

"Sputnik" is a dsDNA virus, referred to as a virophage, that is coassembled with Mimivirus in the host amoeba. We have used cryo-EM to produce an electron density map of the icosahedral Sputnik virus at 3.5-Å resolution, sufficient to verify the identity of most amino acids in the capsid proteins and to establish the identity of the pentameric protein forming the fivefold vertices. It was also shown that the virus lacks an internal membrane. The capsid is organized into a T = 27 lattice in which there are 260 trimeric capsomers and 12 pentameric capsomers. The trimeric capsomers consist of three double "jelly-roll" major capsid proteins creating pseudohexameric capsomer symmetry. The pentameric capsomers consist of five single jelly-roll proteins. The release of the genome by displacing one or more of the pentameric capsomers may be the result of a low-pH environment. These results suggest a mechanism of Sputnik DNA ejection that probably also occurs in other big icosahedral double jelly-roll viruses such as Adenovirus. In this study, the near-atomic resolution structure of a virus has been established where crystallization for X-ray crystallography was not feasible.

Structure and function of the small terminase component of the DNA packaging machine in T4-like bacteriophages.

Tailed DNA bacteriophages assemble empty procapsids that are subsequently filled with the viral genome by means of a DNA packaging machine situated at a special fivefold vertex. The packaging machine consists of a "small terminase" and a "large terminase" component. One of the functions of the small terminase is to initiate packaging of the viral genome, whereas the large terminase is responsible for the ATP-powered translocation of DNA. The small terminase subunit has three domains, an N-terminal DNA-binding domain, a central oligomerization domain, and a C-terminal domain for interacting with the large terminase. Here we report structures of the central domain in two different oligomerization states for a small terminase from the T4 family of phages. In addition, we report biochemical studies that establish the function for each of the small terminase domains. On the basis of the structural and biochemical information, we propose a model for DNA packaging initiation.

Spontaneous Regression of a Large Vestibular Schwannoma: Is Nonoperative Management Reasonable?

Vestibular schwannomas (VSs) are the most common cerebellopontine tumors. The natural history of smaller-sized VSs (<30 mm) has been well-studied, leading to the recommendation of a "watch and wait" approach. However, large VSs (>30 mm) have not been extensively studied, mainly because of their rarity. As such, most patients are conventionally offered surgery which carries a significant risk of neurological morbidity. Here, we report a case of a giant VS (>40 mm) in a 30-year-old man who regressed spontaneously. He was lost to follow-up for 18 years and, upon re-presentation, the symptomatology drastically improved and repeat imaging demonstrated a marked reduction in tumor size. Referring to similar cases in other studies, we postulate that most large and giant VSs undergo a phase of growth and stasis, followed by regression due to shifts in the balance between tumorigenic and regressive factors. Taken together with emerging molecular data, further studies are required to better understand the history of large and giant VSs to shape more personalized treatment options. This potentially includes non-operative management as a tenable option.

Ameliorative effects of the Coptis inflorescence extract against lung injury in diabetic mice by regulating AMPK/NEU1 signaling.

BACKGROUND: In diabetic patients, complications are the leading cause of death and disability, while diabetic lung damage has received little research. The Coptis inflorescence extract (CE) has hypoglycemic properties, but the mechanism of its protective role on diabetic lung injury is understood. PURPOSE: This study aims to explore the protective actions and molecular mechanism of CE and its active ingredients in diabetic lung disease. METHOD: Twenty-nine metabolites were identified in the metabolomic profile of CE using HPLC-ESI/MS, and high-content substances of berberine (BBR) and linarin (LIN) were isolated from CE using column chromatography. The potential targets and molecular mechanisms of CE against diabetic lung damage were systematically investigated by network pharmacology and in vitro experimental validation. RESULTS: CE significantly improved lung function and pathology. CE (360 mg/kg) or metformin treatment significantly improved lipid metabolism disorders, including decreased HDL-C and elevated serum TG, TC, and LDL-C levels. Furthermore, CE's chemical composition was determined using the HPLC-QTOF-MS method. CE identified five compounds as candidate active compounds (Berberine, Linarin, Palmatine, Worenine, and Coptisine). Network pharmacology analysis predicted CE contained five active compounds and target proteins, that AMPK, TGFß1, and Smad might be the key targets in treating diabetic lung injury. Then we investigated the therapeutic effect of bioactive compounds of CE on diabetic lung damage through in vivo and in vitro experiments. Intragastric administration with BBR (50 mg/kg) or LIN (20 mg/kg) suppressed weight loss, hyperglycemia, and dyslipidemia, significantly alleviating lung inflammation in diabetic mice. Further mechanism research revealed that LIN or BBR inhibited alveolar epithelial-mesenchymal transition induced by high glucose by regulating AMPK/NEU-mediated signaling pathway. CONCLUSION: In conclusion, the administration of CE can effectively alleviate diabetic lung damage, providing a scientific basis for lowering blood sugar to moisturize lung function. BBR and LIN, the main components of CE, can effectively alleviate diabetic lung damage by regulating AMPK/NEU1 Signaling and inhibiting the TGF-ß1 level, which may be a critical mechanism of its effects.

Epidemiological Insights into the Omicron Outbreak via MeltArray-Assisted Real-Time Tracking of SARS-CoV-2 Variants.

The prolonged course of the COVID-19 pandemic necessitates sustained surveillance of emerging variants. This study aimed to develop a multiplex real-time polymerase chain reaction (rt-PCR) suitable for the real-time tracking of Omicron subvariants in clinical and wastewater samples. Plasmids containing variant-specific mutations were used to develop a MeltArray assay. After a comprehensive evaluation of both analytical and clinical performance, the established assay was used to detect Omicron variants in clinical and wastewater samples, and the results were compared with those of next-generation sequencing (NGS) and droplet digital PCR (ddPCR). The MeltArray assay identified 14 variant-specific mutations, enabling the detection of five Omicron sublineages (BA.2*, BA.5.2*, BA.2.75*, BQ.1*, and XBB.1*) and eight subvariants (BF.7, BN.1, BR.2, BQ.1.1, XBB.1.5, XBB.1.16, XBB.1.9, and BA.4.6). The limit of detection (LOD) of the assay was 50 copies/reaction, and no cross-reactivity was observed with 15 other respiratory viruses. Using NGS as the reference method, the clinical evaluation of 232 swab samples exhibited a clinical sensitivity of > 95.12% (95% CI 89.77-97.75%) and a specificity of > 95.21% (95% CI, 91.15-97.46%). When used to evaluate the Omicron outbreak from late 2022 to early 2023, the MeltArray assay performed on 1408 samples revealed that the epidemic was driven by BA.5.2* (883, 62.71%) and BF.7 (525, 37.29%). Additionally, the MeltArray assay demonstrated potential for estimating variant abundance in wastewater samples. The MeltArray assay is a rapid and scalable method for identifying SARS-CoV-2 variants. Integrating this approach with NGS and ddPCR will improve variant surveillance capabilities and ensure preparedness for future variants.

Structural studies of the giant mimivirus.

Mimivirus is the largest known virus whose genome and physical size are comparable to some small bacteria, blurring the boundary between a virus and a cell. Structural studies of Mimivirus have been difficult because of its size and long surface fibers. Here we report the use of enzymatic digestions to remove the surface fibers of Mimivirus in order to expose the surface of the viral capsid. Cryo-electron microscopy (cryoEM) and atomic force microscopy were able to show that the 20 icosahedral faces of Mimivirus capsids have hexagonal arrays of depressions. Each depression is surrounded by six trimeric capsomers that are similar in structure to those in many other large, icosahedral double-stranded DNA viruses. Whereas in most viruses these capsomers are hexagonally close-packed with the same orientation in each face, in Mimivirus there are vacancies at the systematic depressions with neighboring capsomers differing in orientation by 60 degrees . The previously observed starfish-shaped feature is well-resolved and found to be on each virus particle and is associated with a special pentameric vertex. The arms of the starfish fit into the gaps between the five faces surrounding the unique vertex, acting as a seal. Furthermore, the enveloped nucleocapsid is accurately positioned and oriented within the capsid with a concave surface facing the unique vertex. Thus, the starfish-shaped feature and the organization of the nucleocapsid might regulate the delivery of the genome to the host. The structure of Mimivirus, as well as the various fiber components observed in the virus, suggests that the Mimivirus genome includes genes derived from both eukaryotic and prokaryotic organisms. The three-dimensional cryoEM reconstruction reported here is of a virus with a volume that is one order of magnitude larger than any previously reported molecular assembly studied at a resolution of equal to or better than 65 Angstroms.

Giant Marseillevirus highlights the role of amoebae as a melting pot in emergence of chimeric microorganisms.

Giant viruses such as Mimivirus isolated from amoeba found in aquatic habitats show biological sophistication comparable to that of simple cellular life forms and seem to evolve by similar mechanisms, including extensive gene duplication and horizontal gene transfer (HGT), possibly in part through a viral parasite, the virophage. We report here the isolation of "Marseille" virus, a previously uncharacterized giant virus of amoeba. The virions of Marseillevirus encompass a 368-kb genome, a minimum of 49 proteins, and some messenger RNAs. Phylogenetic analysis of core genes indicates that Marseillevirus is the prototype of a family of nucleocytoplasmic large DNA viruses (NCLDV) of eukaryotes. The genome repertoire of the virus is composed of typical NCLDV core genes and genes apparently obtained from eukaryotic hosts and their parasites or symbionts, both bacterial and viral. We propose that amoebae are "melting pots" of microbial evolution where diverse forms emerge, including giant viruses with complex gene repertoires of various origins.

Damage law and mechanism of coal-rock joint structure induced by liquid nitrogen at low temperature.

The width and degree of connectivity of coal-rock joints directly affect the seepage capacity of flow energy such as gas. To study the damage law and mechanism of the coal-rock joint structure under the action of liquid nitrogen, two methods of liquid nitrogen unloaded and liquid nitrogen freeze-thaw were used to carry out damage modification experiments on coal-rock with different water saturation. Using OLS4000 laser confocal microscope and MH-25 universal testing machine to conduct electron microscope scanning and uniaxial compression test, measure the joint width expansions and Young's modulus of the coal-rock surface before and after the test, establish a physical and mechanical model of freeze-thaw damage to analyze the ice-wedge expansion stress influence on the damage of coal-rock joint structure and establish damage criterion. The research results show that the ice-wedge expansion stress, confining pressure, and temperature stress in the joint jointly affect the structural damage of coal-rock joints, and the ice-wedge expansion stress contributes the most. With the increase of water saturation, the damage to the coal-rock joint structure intensifies, and the ice-wedge expansion stress under the water saturation state has the most obvious influence on the damage to the coal-rock joint structure. The damage criterion constructed by the freeze-thaw damage physical-mechanical model can reveal the damage mechanism of the effect of ice-wedge expansion stress on the coal-rock joint structure. This paper has certain practical significance for the safety and stability evaluation of rock engineering in cold and arid regions and provides new ideas for effectively extracting clean energy such as coalbed methane and preventing rock bursts.

Enhanced degradation mechanism of tetracycline by MnO2 with the presence of organic acids.

In this study, we constructed MnO2/organic acid (OA) systems using MnO2 colloid, the most reactive phase of Mn(IV), and two kinds of OA (oxalic acid and l-tartaric acid). We investigated the effect of OA on tetracycline (TC) degradation by MnO2. The results show that both OA obviously accelerate TC degradation by MnO2. Mn(III) formed during the reaction lead to the acceleration. Mn(III)-oxalate complex formed in oxalic acid system resulted in the lower degradation efficiency than that in l-tartaric acid system. The acceleration of oxalic acid was decreased when the concentration was more than 75 µM, and even completely disappeared with the concentration of 500 µM, owning to the fact that excess oxalic acid decreased the pH and some MnO2 was fast reduced to Mn2+ by oxalic acid and unable to react with TC. The impact of pH on TC degradation resulted from the influences of H+ on MnO2 redox potentials and TC deprotonation. And acidic conditions accelerated TC degradation. The addition of Mg2+, Ca2+, Fe3+ and Zn2+ exhibited an inhibitory effect in both systems for their occupying reactive sites on MnO2 surface and blocking the access of TC to MnO2. Similar intermediates in the two systems were detected, indicating a similar TC degradation mechanism including a series of reactions like dehydration, hydroxylation and oxidation. The MnO2/OA system provides an efficient treatment of TC in wastewater. And it is also noticeable that MnO2/OA system should also have an important effect on the fate of pollutants in environment, from our results.

Structural studies of the Sputnik virophage.

The virophage Sputnik is a satellite virus of the giant mimivirus and is the only satellite virus reported to date whose propagation adversely affects its host virus' production. Genome sequence analysis showed that Sputnik has genes related to viruses infecting all three domains of life. Here, we report structural studies of Sputnik, which show that it is about 740 A in diameter, has a T=27 icosahedral capsid, and has a lipid membrane inside the protein shell. Structural analyses suggest that the major capsid protein of Sputnik is likely to have a double jelly-roll fold, although sequence alignments do not show any detectable similarity with other viral double jelly-roll capsid proteins. Hence, the origin of Sputnik's capsid might have been derived from other viruses prior to its association with mimivirus.

Distinct DNA exit and packaging portals in the virus Acanthamoeba polyphaga mimivirus.

Icosahedral double-stranded DNA viruses use a single portal for genome delivery and packaging. The extensive structural similarity revealed by such portals in diverse viruses, as well as their invariable positioning at a unique icosahedral vertex, led to the consensus that a particular, highly conserved vertex-portal architecture is essential for viral DNA translocations. Here we present an exception to this paradigm by demonstrating that genome delivery and packaging in the virus Acanthamoeba polyphaga mimivirus occur through two distinct portals. By using high-resolution techniques, including electron tomography and cryo-scanning electron microscopy, we show that Mimivirus genome delivery entails a large-scale conformational change of the capsid, whereby five icosahedral faces open up. This opening, which occurs at a unique vertex of the capsid that we coined the "stargate", allows for the formation of a massive membrane conduit through which the viral DNA is released. A transient aperture centered at an icosahedral face distal to the DNA delivery site acts as a non-vertex DNA packaging portal. In conjunction with comparative genomic studies, our observations imply a viral packaging pathway akin to bacterial DNA segregation, which might be shared by diverse internal membrane-containing viruses.

Alignment-free sequence comparison for virus genomes based on location correlation coefficient.

Coronaviruses (especially SARS-CoV-2) are characterized by rapid mutation and wide spread. As these characteristics easily lead to global pandemics, studying the evolutionary relationship between viruses is essential for clinical diagnosis. DNA sequencing has played an important role in evolutionary analysis. Recent alignment-free methods can overcome the problems of traditional alignment-based methods, which consume both time and space. This paper proposes a novel alignment-free method called the correlation coefficient feature vector (CCFV), which defines a correlation measure of the L-step delay of a nucleotide location from its location in the original DNA sequence. The numerical feature is a 16×L-dimensional numerical vector describing the distribution characteristics of the nucleotide positions in a DNA sequence. The proposed L-step delay correlation measure is interestingly related to some types of L+1 spaced mers. Unlike traditional gene comparison, our method avoids the computational complexity of multiple sequence alignment, and hence improves the speed of sequence comparison. Our method is applied to evolutionary analysis of the common human viruses including SARS-CoV-2, Dengue virus, Hepatitis B virus, and human rhinovirus and achieves the same or even better results than alignment-based methods. Especially for SARS-CoV-2, our method also confirms that bats are potential intermediate hosts of SARS-CoV-2.

Photodegradation of dissolved organic matter of chicken manure: Property changes and effects on Zn2+/Cu2+ binding property.

Untreated livestock manure contains high concentrations of dissolved organic matter (DOM), which can enter the environment through leaching and eluviation, showing an important impact on the environment. In this research, fresh chicken manure from a large-scale chicken farm was collected as the source of DOM. The infrared spectrum of the original DOM was characterized. TOC analysis, UV spectrum and 3D fluorescence spectrum were used to measure the properties of DOM before and after photodegradation. Infrared spectroscopy results show that chicken manure DOM may contain aliphatic and aromatic compounds, alcohols, phenols, polysaccharides and some protein substances; In three systems, the order of TOC removal rates of DOM was water + UV system (85%) > > water + simulated sunlight system (7.2%) > ice + simulated sunlight system (4.5%); Changes of UV spectra, fluorescence spectra, molecular weight distribution and pH value show that, in three systems, as the illumination time increased, photodegradation reduced pH value of the systems, aromaticity and humus contents of DOM, while increased the proportion of medium and/or small molecular weight components of DOM. The amounts of all these changes were proportional to DOM photodegradation rates in the system. The binding ability of DOM with Cu2+ and Zn2+ in water solution decreased significantly after the photodegradation.

The three-dimensional structure of Mimivirus.

Mimivirus, the prototypic member of the new family of Mimiviridae, is the largest virus known to date. Progress has been made recently in determining the three-dimensional structure of the 0.75-microm diameter virion using cryo-electron microscopy and atomic force microscopy. These showed that the virus is composed of an outer layer of dense fibers surrounding an icosahedrally shaped capsid and an internal membrane sac enveloping the genomic material of the virus. Additionally, a unique starfish-like structure at one of the fivefold vertices, required by the virus for infecting its host, has been defined in more detail.

The headful packaging nuclease of bacteriophage T4.

Most tailed bacteriophages and herpes viruses replicate genome as a concatemer which is cut by a 'headful' nuclease upon completion of genome packaging. Here, the catalytic centre of phage T4 headful nuclease, present in the C-terminal domain of 'large terminase' gp17, has been defined by mutational, biochemical and structural analyses. The crystal structure shows that this nuclease has an RNase-H fold, suggesting that it cuts DNA by a two-metal ion mechanism. The active centre has a Mg ion co-ordinated by three acidic residues, D401, E458 and D542. Mutations at any of these residues resulted in loss of nuclease activity, but the mutants can package linear DNA. The gp17's nuclease activity is modulated by the 'small terminase', gp16, by the N-terminal ATPase domain of gp17, and by the assembled packaging motor. These results lead to hypotheses concerning how phage headful nucleases cut the viral genomes before and after, but not during, DNA packaging.