Traceability of Microplastic Fragments from Waste Plastic Express Packages Using Near-Infrared Spectroscopy Combined with Chemometrics.

The pollution from waste plastic express packages (WPEPs), especially microplastic (MP) fragments, caused by the blowout development of the express delivery industry has attracted widespread attention. On account of the variety of additives, strong complexity, and high diversity of plastic express packages (PEPs), the multi-class classification of WPEPs is a typical large-class-number classification (LCNC). The traceability and identification of microplastic fragments from WPEPs is very challenging. An effective chemometric method for large-class-number classification would be very beneficial for the comprehensive treatment of WPEP pollution through the recycling and reuse of waste plastic express packages, including microplastic fragments and plastic debris. Rather than using the traditional one-against-one (OAO) and one-against-all (OAA) dichotomies, an exhaustive and parallel half-against-half (EPHAH) decomposition, which overcomes the defects of the OAO's classifier learning limitations and the OAA's data proportion imbalance, is proposed for feature selection. EPHAH analysis, combined with partial least squares discriminant analysis (PLS-DA) for large-class-number classification, was performed on 750 microplastic fragments of polyethylene WPEPs from 10 major courier companies using near-infrared (NIR) spectroscopy. After the removal of abnormal samples through robust principal component analysis (RPCA), the root mean square error of cross-validation (RMSECV) value for the model was reduced to 0.01, which was 21.5% lower than that including the abnormal samples. The best models of PLS-DA were obtained using SNV combined with SG-17 smoothing and 2D (SNV+SG-17+2D); the latent variables (LVs), the error rates of Monte Carlo cross-validation (ERMCCVs), and the final classification accuracies were 6.35, 0.155, and 88.67% for OAO-PLSDA; 5.37, 0.103, and 87.33% for OAA-PLSDA; and 3.12, 0.054, and 96.00% for EPHAH-PLSDA. The results showed that the EPHAH strategy can completely learn the complex LCNC decision boundaries for 10 classes, effectively break the tie problem, and greatly improve the voting resolution, thereby demonstrating significant superiority to both the OAO and OAA strategies in terms of classification accuracy. Meanwhile, PLS-DA further maximized the covariance and data interpretation abilities between the potential variables and categories of microplastic debris, thereby establishing an ideal performance identification model with a recognition rate of 96.00%.

Remediation of uranium-contaminated alkaline soil by rational application of phosphorus fertilizers: Effect and mechanism.

In alkaline soil, abundant carbonates will mobilize uranium (U) and increase its ecotoxicity, which is a serious threat to crop growth. However, the knowledge of U remediation in alkaline soils remains very limited. In this study, U-contaminated alkaline soil (tillage layer) was collected from the Ili mining area of Xinjiang, the soil remediation was carried out by using phosphorus (P) fertilizers of different solubility (including KH2PO4, Ca(H2PO4)2, CaHPO4, and Ca3(PO4)2), and the pathways and mechanisms of U passivation in the alkaline soil were revealed. The results showed that water-soluble P fertilizers, KH2PO4 and Ca(H2PO4)2, were highly effective at immobilizing U, and significantly reduced the bioavailability of soil U. The exchangeable U was reduced by 70.5 ± 0.1% (KH2PO4) and 68.2 ± 1.9% (Ca(H2PO4)2), which was converted into the Fe-Mn oxide-bound and residual phases. Pot experiments showed that soil remediation by KH2PO4 significantly promoted crop growth, especially for roots, and reduced U uptake in crops by 94.5 ± 1.0%. The immobilization of U by KH2PO4 could be attributed to the release of phosphate anions, which react with the uranyl ion (UO22+) forming a stable mineral of meta-ankoleite and enhancing the binding of UO22+ to the soil Fe-Mn oxides. In addition, KH2PO4 dissolution produces acidity and P fertilizer, which can reduce soil alkalinity and improve crop growth. The findings in this work demonstrate that a rational application of P fertilizer can effectively, conveniently, and cheaply remediate U contamination and improve crop yield and safety on alkaline farmland.

Diurnal temperature variations in the lower troposphere as measured by an all-day-operational pure rotational Raman lidar.

We describe a pure rotational Raman lidar for measuring the all-day temperature profiles in the lower troposphere. The lidar is made up of a frequency-tripled Nd:YAG laser at 354.82 nm with ∼250mJ pulse energy at the 30 Hz repetition rate, a 200 mm receiving telescope, and narrow-band interference-filter-based detection optics. The lidar performance is shown by measured examples. Under clear sky conditions, with an integration time of 60 min and a vertical resolution of 90 m, the 1-σ statistical uncertainty does not exceed 1 K up to the altitude of ∼4.1km during nighttime, while the corresponding altitude is ∼2.3km at noon. The diurnal temperature variation characteristics have been revealed by the lidar measurements with the 1-σ statistical uncertainty <1K between altitudes ranging from 0.6 to ∼2.0km at Wuhan, China (30.53°N, 114.37°E). The atmospheric temperature shows a strong diurnal oscillation and moderate semidiurnal oscillation at altitudes 0-1.4 km for two days in July 2019 (July period), 0-1.4 km for four days in September 2019 (September period), and 0-0.8 km for three days in January 2018 (January period), respectively. The mean diurnal and semidiurnal amplitudes of the nine days are respectively ∼1.4K and ∼0.5K at the 0.6 km altitude, while the corresponding surface values are ∼4.2K and ∼1.4K, respectively. The diurnal amplitudes tend to weaken with increasing altitude. At altitudes >0.6km, the diurnal amplitude in the September period is less than that in the July period, but greater than that in the January period. The phase delays of the diurnal oscillations are ∼3h in the July period, 5-6 h in the September period, and 5-7 h in the January period compared to those at the surface, respectively. Both the diurnal amplitudes and phase delays indicate a possible seasonal dependence.

Enantioselective effects of imazethapyr residues on Arabidopsis thaliana metabolic profile and phyllosphere microbial communities.

Imazethapyr (IM) is a widely used acetolactate synthase-inhibiting chiral herbicide. It has long-term residuals that may be absorbed by the human body through the edible parts of plants, such as vegetable leaves or fruits. Here, we selected a model plant, Arabidopsis thaliana, to determine the effects of R-IM and S-IM on its leaf structure, photosynthetic efficiency, and metabolites, as well as the structures of microorganisms in the phyllosphere, after 7 days of exposure. Our results indicated enantiomeric differences in plant growth between R-IM and S-IM; 133 µg/kg R-IM showed heavier inhibition of photosynthetic efficiency and greater changes to subcellular structure than S-IM. R-IM and S-IM also had different effects on metabolism and leaf microorganisms. S-IM mainly increased lipid compounds and decreased amino acids, while R-IM increased sugar accumulation. The relative abundance of Moraxellaceae human pathogenic bacteria was increased by R-IM treatment, indicating that R-IM treatment may increase leaf surface pathogenic bacteria. Our research provides a new perspective for evaluating the harmfulness of pesticide residues in soil, phyllosphere microbiome changes via the regulation of plant metabolism, and induced pathogenic bacterial accumulation risks.

Optimizing sludge dewatering with a combined conditioner of Fenton's reagent and cationic surfactant.

Enhancing sludge dewatering is of importance in reducing environmental burden and disposal costs. In this work, a cationic surfactant, cetyl trimethyl ammonium bromide (CTAB), was combined with Fenton's reagent for sludge dewatering. Results show that the Fenton-CTAB conditioning significantly promotes the sludge dewatering. Using combined techniques of response surface methodology and uniform design, dosages of Fe2+, H2O2, and CTAB for water content response were optimized to be 89, 276, and 233 mg/g dry solids (DS), respectively. The water content of sludge decreased from 79.0% to 66.8% under the optimal conditions. Compared with cationic polyacrylamide, the Fenton-CTAB system exhibited superior sludge dewatering performance. To gain insights into the mechanisms involved in sludge dewatering, the effects of Fenton-CTAB conditioning on the composition of extracellular polymeric substances (EPS) and the morphology of the sludge flocs were investigated. The decomposition of EPS into some dissolved organics and the release of proteins in tightly bound EPS facilitated the conversion of bound water to free water and further reduced the water content of sludge cake. After conditioning, morphology of sludge showed aggregation. Overall, the enhanced sludge dewatering by Fenton-CTAB treatment provides an efficient way for management of sewage sludge.

Fabricating biogenic Fe(III) flocs from municipal sewage sludge using NAFO processes: Characterization and arsenic removal ability.

This study involved fabricating biogenic Fe(III) flocs enriched from municipal sludge using microbial nitrate-dependent anaerobic Fe(II)-oxidizing (NAFO) processes. The research focused on bacterial community compositions and physicochemical properties of the biogenic Fe(III) flocs and their ability to adsorb arsenic (As). High-throughput sequencing analysis showed that significant microbial succession occurs in the raw sludge after the NAFO processes. The predominant bacterial communities in the biogenic Fe(III) flocs included Rhodanobacter, Parvibaculum, Gemmatimonas and Segetibacter genera. Microscopic and spectroscopic analyses included scanning electron microscopy - energy disperse spectroscopy (SEM-EDS), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy. These tests indicated that biogenic Fe(III) flocs were a mixture of NAFO bacteria and nanosized, poorly crystalline Fe(III) oxide particles. Batch experiments showed that after 120 min of reaction time, more than 95% of As(III) and As(V) (at an initial concentrations of 0.25 mg/L) were effectively removed with 120 ppm biogenic Fe(III) flocs. In addition, biogenic Fe(III) flocs removed As more effectively than abiotic Fe(III) flocs. These findings indicated that biogenic Fe(III) flocs produced from municipal sludge using NAFO processes performed well in removing As.

Effects of imazethapyr spraying on plant growth and leaf surface microbial communities in Arabidopsis thaliana.

Imazethapyr (IM) is an acetolactate synthase (ALS)-inhibiting herbicide that has been widely used in recent years. However, IM spraying can lead to the accumulation of herbicide residues in leaves. Here, we determined the effects of IM spraying on the plant growth and leaf surface microbial communities of Arabidopsis thaliana after 7 and 14 days of exposure. The results suggested that IM spraying inhibited plant growth. Fresh weight decreased to 48% and 26% of the control value after 7 and 14 days, respectively, of 0.035 kg/ha IM exposure. In addition, anthocyanin content increased 9.2-fold and 37.2-fold relative to the control content after 7 and 14 days of treatment, respectively. Furthermore, IM spraying destroyed the cell structures of the leaves, as evidenced by increases in the number of starch granules and the stomatal closure rate. Reductions in photosynthetic efficiency and antioxidant enzyme activity were observed after IM spraying, especially after 14 days of exposure. The diversity and evenness of the leaf microbiota were not affected by IM treatment, but the composition of community structure at the genus level was altered by IM spraying. Imazethapyr application increased the abundance of Pseudomonas, a genus that includes species pathogenic to plants and humans, indicating that IM potentially increased the abundance of pathogenic bacteria on leaves. Our findings increase our understanding of the relationships between herbicide application and the microbial community structures on plant leaves, and they provide a new perspective for studying the ecological safety of herbicide usage.

Single-line-extracted pure rotational Raman lidar to measure atmospheric temperature and aerosol profiles.

We have built a pure rotational Raman (PRR) lidar system that effectively detects two isolated N2 molecule PRR line signals and elastic backscatter signals. This system enables all-day temperature profiles to be accurately obtained without calibration, according to the simple two-parameter functional relationship between the temperature and ratio of the two PRR line signals. Based on the derived temperature profiles, the aerosol backscatter and extinction profiles can be further determined strictly from one measured PRR line signal and elastic backscatter signal without additional assumptions. The two aerosol parameters and resultant lidar ratio provide strict standards for the lidar measurements of aerosol.

Ecological Effects of Single-Walled Carbon Nanotubes on Soil Microbial Communities and Soil Fertility.

The manufacturers of single-walled carbon nanotubes (SWCNTs) are continuously expanding their manufacturing and commercial markets, indicating that the environmental release and accumulation of SWCNTs in soil is inevitable. However, little is known about the effects of SWCNTs on soil physicochemical properties and soil microbial communities. Our results showed that treatment with SWCNTs resulted in an enhancement of microorganism metabolism related to soil organic compound degradation and a change in the structure of soil microbial communities, but the diversity of soil microorganisms was not significantly affected. The decrease in soil urease activity and the increase in the relative abundance of Nitrospirae after SWCNTs exposure might be relevant to the induction of soil nitrification. The relative abundances of phosphate-solubilizing microorganisms increased after exposure to SWCNTs, which was beneficial for phosphorus bioavailability in the soil. Our current study highlights that exposure to SWCNTs at concentrations of 3 and 10 µg/g can change the composition of soil microorganism communities, promote soil organic degradation and improve soil fertility by enhancing N and P availability in a short time.

Uranium Bioreduction and Biomineralization.

Following the development of nuclear science and technology, uranium contamination has been an ever increasing concern worldwide because of its potential for migration from the waste repositories and long-term contaminated environments. Physical and chemical techniques for uranium pollution are expensive and challenging. An alternative to these technologies is microbially mediated uranium bioremediation in contaminated water and soil environments due to its reduced cost and environmental friendliness. To date, four basic mechanisms of uranium bioremediation-uranium bioreduction, biosorption, biomineralization, and bioaccumulation-have been established, of which uranium bioreduction and biomineralization have been studied extensively. The objective of this review is to provide an understanding of recent developments in these two fields in relation to relevant microorganisms, mechanisms, influential factors, and obstacles.

Spatial Variability of Cyanobacteria and Heterotrophic Bacteria in Lake Taihu (China).

Cyanobacterial blooms frequently occur in Lake Taihu (China), but the intertwined relationships between biotic and abiotic factors modulating the frequency and duration of the blooms remain enigmatic. To better understand the relationships between the key abiotic and biotic factors and cyanobacterial blooms, we measured the abundance and diversity of prokaryotic organisms by high-throughput sequencing, the abundance of key genes involved in microcystin production and nitrogen fixation or loss as well as several physicochemical parameters at several stations in Lake Taihu during a cyanobacterial bloom of Microcystis sp.. Measurements of the copy number of denitrification-related genes and 16S rRNA analyses show that denitrification potential and denitrifying bacteria abundance increased in concert with non-diazotrophic cyanobacteria (Microcystis sp.), suggesting limited competition between cyanobacteria and heterotrophic denitrifiers for nutrients, although potential bacteria-mediated N loss may hamper Microcystis growth. The present study provides insight into the importance of different abiotic and biotic factors in controlling cyanobacteria and heterotrophic bacteria spatial variability in Lake Taihu.

Microbially-induced Carbonate Precipitation for Immobilization of Toxic Metals.

Rapid urbanization and industrialization resulting from growing populations contribute to environmental pollution by toxic metals and radionuclides which pose a threat to the environment and to human health. To combat this threat, it is important to develop remediation technologies based on natural processes that are sustainable. In recent years, a biomineralization process involving ureolytic microorganisms that leads to calcium carbonate precipitation has been found to be effective in immobilizing toxic metal pollutants. The advantage of using ureolytic organisms for bioremediating metal pollution in soil is their ability to immobilize toxic metals efficiently by precipitation or coprecipitation, independent of metal valence state and toxicity and the redox potential. This review summarizes current understanding of the ability of ureolytic microorganisms for carbonate biomineralization and applications of this process for toxic metal bioremediation. Microbial metal carbonate precipitation may also be relevant to detoxification of contaminated process streams and effluents as well as the production of novel carbonate biominerals and biorecovery of metals and radionuclides that form insoluble carbonates.

Effect of Metal Ions on the Formation of Trichloronitromethane during Chlorination of Catechol and Nitrite.

Catechol, nitrite, and dissolved metals are ubiquitous in source drinking water. Catechol and nitrite have been identified as precursors for halonitromethanes (HNMs), but the effect of metal ions on HNM formation during chlorination remains unclear. The main objective of this study was to investigate the effect of metal ions (Fe, Ti, Al) on the formation of trichloronitromethane (TCNM) (the most representative HNM species in disinfected water) on chlorinating catechol and nitrite. Trichloronitromethane was extracted by methyl tert-butyl ether and detected by gas chromatography. The results show that metal ions promoted the formation of TCNM and that the enhancement efficiency followed the order of Fe > Ti > Al. Trichloronitromethane formation increased greatly within 2 h, and a basic condition (pH 8-9) favored TCNM formation more than acidic or neutral conditions. The conjoint effect of the metal-ion mixtures was shown to be similar to that of the single metal ion having the highest promoting effect on TCNM formation. Our results strongly suggest that metal ions play a significant role in enhancing TCNM formation.

Analysis of the Proteome of the Marine Diatom Phaeodactylum tricornutum Exposed to Aluminum Providing Insights into Aluminum Toxicity Mechanisms.

Trace aluminum (Al) concentrations can be toxic to marine phytoplankton, the basis of the marine food web, but the fundamental Al toxicity and detoxification mechanisms at the molecular levels are poorly understood. Using an array of proteomic, transcriptomic, and biochemical techniques, we describe in detail the cellular response of the model marine diatom Phaeodactylum tricornutum to a short-term sublethal Al stress (4 h of exposure to 200 µM total initial Al). A total of 2204 proteins were identified and quantified by isobaric tags for relative and absolute quantification (iTRAQ) in response to the Al stress. Among them, 87 and 78 proteins performing various cell functions were up- and down-regulated after Al treatment, respectively. We found that photosynthesis was a key Al toxicity target. The Al-induced decrease in electron transport rates in thylakoid membranes lead to an increase in reactive oxygen species (ROS) production, which cause increased lipid peroxidation. Several ROS-detoxifying proteins were induced to help decrease Al-induced oxidative stress. In parallel, glycolysis and pentose phosphate pathway were up-regulated in order to produce cell energy (NADPH, ATP) and carbon skeleton for cell growth, partially circumventing the Al-induced toxicity effects on photosynthesis. These cellular responses to Al stress were coordinated by the activation of various signal transduction pathways. The identification of Al-responsive proteins in the model marine phytoplankton P. tricornutum provides new insights on Al stress responses as well as a good start for further exploring Al detoxification mechanisms.

PSI showed higher tolerance to Sb(V) than PSII due to stimulation of cyclic electron flow around PSI.

The knowledge of the effects of Sb(V) on the physiological characteristics of cyanobacteria was still limited. In the present study, responses of photosystem I and II (PSI and PSII), cyclic electron flow (CEF), and interphotosystem electron transport of Microcystis aeruginosa to 5-100 mg/l Sb(V) were synchronously measured using the Dual-PAM-100. 5 mg/l Sb (V) significantly inhibited PSII activity, but had no significant effects on PSI activity. At higher concentrations of Sb(V), the quantum yield and electron transport of PSI were less affected compared to PSII. The ratio of Y(II)/Y(I) significantly decreased with increasing Sb(V) concentration. It decreased from 0.7 for control to 0.4 for 100 mg/l Sb(V)-treated cells, indicating that the change of the distribution of quantum yields between two photosystems and more serious inhibition of PSII under stress of Sb(V) compared to PSI. CEF was activated associated with the inhibition of linear electron flow after exposure to Sb(V). The contribution of Y(CEF) to the quantum yield and activity of PSI increased with increasing Sb(V) concentrations. The cyclic electron transport rate made a significant contribution to electron transport rate of PSI, especially at high Sb(V) concentration (100 mg/l) and high illumination (above 555 µmol photons/m(2)/s). The stimulation of CEF was essential for the higher tolerance of PSI than PSII to Sb(V).

Herbicidal effects of harmaline from Peganum harmala on photosynthesis of Chlorella pyrenoidosa: probed by chlorophyll fluorescence and thermoluminescence.

The herbicidal effects of harmaline extracted from Peganum harmala seed on cell growth and photosynthesis of green algae Chlorella pyrenoidosa were investigated using chlorophyll a fluorescence and thermoluminescence techniques. Exposure to harmaline inhibited cell growth, pigments contents and oxygen evolution of C. pyrenoidosa. Oxygen evolution was more sensitive to harmaline toxicity than cell growth or the whole photosystem II (PSII) activity, maybe it was the first target site of harmaline. The JIP-test parameters showed that harmaline inhibited the donor side of PSII. Harmaline decreased photochemical efficiency and electron transport flow of PSII but increased the energy dissipation. The charge recombination was also affected by harmaline. Amplitude of the fast phase decreased and the slow phase increased at the highest level of harmaline. Electron transfer from QA(-) to QB was inhibited and backward electron transport flow from QA(-) to oxygen evolution complex was enhanced at 10 µg mL(-1) harmaline. Exposure to 10 µg mL(-1) harmaline caused appearance of C band in thermoluminescence. Exposure to 5 µg mL(-1) harmaline inhibited the formation of proton gradient. The highest concentration of harmaline treatment inhibited S3QB(-) charge recombination but promoted formation of QA(-)YD(+) charge pairs. P. harmala harmaline may be a promising herbicide because of its inhibition of cell growth, pigments synthesis, oxygen evolution and PSII activities.

A systematic review of antibiotic resistance driven by metal-based nanoparticles: Mechanisms and a call for risk mitigation.

Elevations in antibiotic resistance genes (ARGs) are due not only to the antibiotic burden, but also to numerous environmental pressures (e.g., pesticides, metal ions, or psychotropic pharmaceuticals), which have led to an international public health emergency. Metal-based nanoparticles (MNPs) poison bacteria while propelling nanoresistance at ambient or sub-lethal concentrations, acting as a wide spectrum germicidal agent. Awareness of MNPs driven antibiotic resistance has created a surge of investigation into the molecule mechanisms of evolving and spreading environmental antibiotic resistome. Co-occurrence of MNPs resistance and antibiotic resistance emerge in environmental pathogens and benign microbes may entail a crucial outcome for human health. In this review we expound on the systematic mechanism of ARGs proliferation under the stress of MNPs, including reactive oxygen species (ROS) induced mutation, horizontal gene transfer (HGT) relevant genes regulation, nano-property, quorum sensing, and biofilm formation and highlighting on the momentous contribution of nanoparticle released ion. As antibiotic resistance pattern alteration is closely knit with the mediate activation of nanoparticle in water, soil, manure, or sludge habitats, we have proposed a virulence and evolution based antibiotic resistance risk assessment strategy for MNP contaminated areas and discussed practicable approaches that call for risk management in critical environmental compartments.

How micro-/nano-plastics influence the horizontal transfer of antibiotic resistance genes - A review.

Plastic debris such as microplastics (MPs) and nanoplastics (NPTs), along with antibiotic resistance genes (ARGs), are pervasive in the environment and are recognized as significant global health and ecological concerns. Micro-/nano-plastics (MNPs) have been demonstrated to favor the spread of ARGs by enhancing the frequency of horizontal gene transfer (HGT) through various pathways. This paper comprehensively and systematically reviews the current study with focus on the influence of plastics on the HGT of ARGs. The critical role of MNPs in the HGT of ARGs has been well illustrated in sewage sludge, livestock farms, constructed wetlands and landfill leachate. A summary of the performed HGT assay and the underlying mechanism of plastic-mediated transfer of ARGs is presented in the paper. MNPs could facilitate or inhibit HGT of ARGs, and their effects depend on the type, size, and concentration. This review provides a comprehensive insight into the effects of MNPs on the HGT of ARGs, and offers suggestions for further study. Further research should attempt to develop a standard HGT assay and focus on investigating the impact of different plastics, including the oligomers they released, under real environmental conditions on the HGT of ARGs.

Photochemical demethylation of methylmercury (MeHg) in aquatic systems: A review of MeHg species, mechanisms, and influencing factors.

Photodemethylation is the major pathway of methylmercury (MeHg) demethylation in surface water before uptake by the food chain, whose mechanisms and influence factors are still not completely understood. Here, we review the current knowledge on photodemethylation of MeHg and divide MeHg photolysis into three pathways: (1) direct photodemethylation, (2) free radical attack, and (3) intramolecular electron or energy transfer. In aquatic environments, dissolved organic matter is involved into all above pathways, and due to its complex compositions, properties and concentrations, DOM poses multiple functions during the PD of MeHg. DOM-MeHg complex (mainly by sulfur-containing molecules) might weaken the C-Hg bond and enhance PD through both direct and indirect pathways. In special, synergistic effects of both strong binding sites and chromophoric moieties in DOM might lead to intramolecular electron or energy transfer. Moreover, DOM might play a role of radical scavenger; while triplet state DOM, which is generated by chromophoric DOM under light, might become a source of free radicals. Apart from DOMs, transition metals, halides, NO3-, NO2-, and carbonates also act as radical initialaters or scavengers, and significantly pose effects on radical demethylation, which is generally mediated by hydroxyl radicals and singlet oxygen. Environmental factors such as pH, light wavelength, light intensity, dissolved oxygen, salinity, and suspended particles also affect the PD of MeHg. This study assessed previously published works on three major mechanisms, with the goal of providing general estimates for photodemethylation under various environment factors according to know effects, and highlighting the current uncertainties for future research directions.