Biochar can be applied to diverse natural and engineered anaerobic systems. Biochar plays biogeochemical roles during its production, storage, and environmental dynamics, one of which is related to the global methane flux governed by methanotrophs and methanogens. Our understanding of relevant mechanisms is currently limited to the roles of biochar in methanotrophic growth, but less is known about the roles of biochar in methanogenic growth. Here, we demonstrated that biochar enhanced the methanogenic growth of a model methanogen, Methanosarcina acetivorans, and the role of biochar as an electron acceptor during methanogenic growth was confirmed, which is referred to as biochar-respiratory growth. The biochar-respiratory growth of M. acetivorans promoted the secretion of extracellular polymeric substances (EPS) with augmented electron transfer capabilities, and the removal of EPS significantly attenuated extracellular electron transfer. Identification and quantification of prosthetic cofactors for EPS suggest an important role of flavin and F420 in extracellular electron transfer. Transcriptomic analysis provided additional insights into the biochar-respiratory growth of M. acetivorans, showing that there was a positive response in transcriptional regulation to the favorable growth environment provided by biochar, which stimulated global methanogenesis. Our results shed more light on the in situ roles of biochar in the ecophysiology of methanogens in diverse anaerobic environments.

Reef corals have been threatened by climate change, with more frequent and intense bleaching events leading to extensive coral mortality and loss of coral cover worldwide. In the face of this, the corals' photosymbiont assemblages have received special attention as a key to better understand the bleaching process and its recovery. To assess the effects of thermal anomalies, the coral Mussismilia harttii and the hydrocoral Millepora alcicornis were monitored through the El Niño 2015/2016 at a Southwestern Atlantic (SWA) coral reef. A severe bleaching event (57% of colonies bleached) was documented, triggered by a < 3 °C-week heatwave, but no mortality was detected. The hydrocoral was more susceptible than the scleractinian, displaying bleaching symptoms earlier and experiencing a longer and more intense bleaching event. The composition of photosymbionts in the M. alcicornis population was affected only at the rare biosphere level (< 5% relative abundance), with the emergence of new symbionts after bleaching. Conversely, a temporary dysbiosis was observed in the M. harttii population, with one of the dominant symbiodiniaceans decreasing in relative abundance at the peak of the bleaching, which negatively affected the total β-diversity. After colonies' complete recovery, symbiodiniaceans' dominances returned to normal levels in both hosts. These results highlight critical differences in how the two coral species cope with bleaching and contribute to the understanding of the role of photosymbionts throughout the bleaching-recovery process.

As one of the low-carbon and high-efficient energy sources, nuclear power is developing vigorously to alleviate the crisis of global climate warming and realize carbon neutrality goals. Meanwhile, the ecological effect of thermal drainage in the nuclear power plant is significantly remarkable, which environmental assessment system has not yet referred to microorganisms. The rapid response of microbial diversity and community structure to environmental changes is crucial for ecosystem stability. This study investigated the bacterial diversity, community construction, and the co-occurrence patterns by 16S rRNA gene amplicon sequencing among gradient warming regions in Tianwan Nuclear Power Plant. The alpha diversity of the high warming region was the lowest in summer, which was dominated by Proteobacteria, whereas the highest bacterial diversity presented in high warming regions in winter, which harbored higher proportions of Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes. The spatial distribution of bacterial communities showed clear separation especially in summer. Strong correlations were between community compositions and environmental factors, such as salinity, DO, TN, and temperature in summer. Furthermore, remarkable seasonality in bacterial co-occurrence patterns was discovered: the robustness of the bacterial co-occurrence network was promoted in winter, while the complexity and robustness were decreased in summer due to the warming of thermal drainage. These findings reveal the potential factors underpinning the influence of thermal drainage on bacterial community structure, which make it possible to predict the ecological effect of the nuclear power plants by exploring how the microbial assembly is likely to respond to the temperature and other environmental changes.

Glyphosate is the most commonly used agricultural herbicide in the world. In aquatic ecosystems, glyphosate often adsorbs to benthic substrates or is metabolized and degraded by microorganisms. The effects of glyphosate on microbial communities vary widely as microorganisms respond differently to exposure. To help understand the impacts of glyphosate on the sediment microbiome, we conducted a microcosm experiment examining the responses of benthic sediment microbial communities to herbicide treatments. Sediments from a prairie pothole wetland were collected, and 16S rRNA gene sequencing was used to analyze community composition 2-h and 14-days after a single treatment of low (0.07 ppm), medium (0.7 ppm), or high (7 ppm) glyphosate, aminomethylphosphonic acid (glyphosate metabolite), or a glyphosate-based commercial formula. We found no significant differences in microbial community composition across treatments, concentration levels, or day of sampling. These findings suggest that microbial species in the Prairie Pothole Region of North America may be tolerant to glyphosate exposure.

The plant's endophytic fungi play an important role in promoting host development and metabolism. Studies have shown that the factors affecting the assembly of the endophyte community mainly include host genotype, vertical transmission, and soil origin. However, we do not know the role of vertically transmitted endohytic fungi influences on the host-plant's endophytic community assembly. Salvia miltiorrhiza from three production areas were used as research objects; we constructed three production area genotypes of S. miltiorrhiza regenerated seedlings simultaneously. Based on high-throughput sequencing, we analyzed the effects of genotype, soil origin, and vertical transmission on endophytic fungal communities. The results show that the community of soil origins significantly affected the endophytic fungal community in the regenerated seedlings of S. miltiorrhiza. The influence of genotype on community composition occurs through a specific mechanism. Genotype may selectively screen certain communities into the seed, thereby exerting selection pressure on the community composition process of offspring. As the number of offspring increases gradually, the microbiota, controlled by genotype and transmitted vertically, stabilizes, ultimately resulting in a significant effect of genotype on community composition.Furthermore, we observed that the taxa influencing the active ingredients are also selected as the vertically transmitted community. Moreover, the absence of an initial vertically transmitted community in S. miltiorrhiza makes it more vulnerable to infection by pathogenic fungi. Therefore, it is crucial to investigate and comprehend the selection model of the vertically transmitted community under varying genotypes and soil conditions. This research holds significant implications for enhancing the quality and yield of medicinal plants and economic crops.

Many insects are associated with endosymbionts that influence the feeding, reproduction, and distribution of their hosts. Although the small green mirid, Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae), a zoophytophagous predator that feeds on plants as well as arthropods, is a globally important biological control agent, its microbiome has not been sufficiently studied. In the present study, we assessed the microbiome variation in 96 N. tenuis individuals from 14 locations throughout Japan, based on amplicon sequencing of the 16S ribosomal RNA gene. Nine major bacteria associated with N. tenuis were identified: Rickettsia, two strains of Wolbachia, Spiroplasma, Providencia, Serratia, Pseudochrobactrum, Lactococcus, and Stenotrophomonas. Additionally, a diagnostic PCR analysis for three typical insect reproductive manipulators, Rickettsia, Wolbachia, and Spiroplasma, was performed on a larger sample size (n = 360) of N. tenuis individuals; the most prevalent symbiont was Rickettsia (69.7%), followed by Wolbachia (39.2%) and Spiroplasma (6.1%). Although some symbionts were co-infected, their prevalence did not exhibit any specific tendency, such as a high frequency in specific infection combinations. The infection frequency of Rickettsia was significantly correlated with latitude and temperature, while that of Wolbachia and Spiroplasma was significantly correlated with host plants. The predominance of these bacteria and the absence of obligate symbionts suggested that the N. tenuis microbiome is typical for predatory arthropods rather than sap-feeding insects. Rickettsia and Wolbachia were vertically transmitted rather than horizontally transmitted from the prey. The functional validation of each symbiont would be warranted to develop N. tenuis as a biological control agent.

Bats harbor diverse intracellular Bartonella bacteria, but there is limited understanding of the factors that influence transmission over time. Investigation of Bartonella dynamics in bats could reveal general factors that control transmission of multiple bat-borne pathogens, including viruses. We used molecular methods to detect Bartonella DNA in paired bat (Pteropus medius) blood and bat flies in the family Nycteribiidae collected from a roost in Faridpur, Bangladesh between September 2020 and January 2021. We detected high prevalence of Bartonella DNA in bat blood (35/55, 64%) and bat flies (59/60, 98%), with sequences grouping into three phylogenetic clades. Prevalence in bat blood increased over the study period (33% to 90%), reflecting an influx of juvenile bats in the population and an increase in the prevalence of bat flies. Discordance between infection status and the clade/genotype of detected Bartonella was also observed in pairs of bats and their flies, providing evidence that bat flies take blood meals from multiple bat hosts. This evidence of bat fly transfer between hosts and the changes in Bartonella prevalence during a period of increasing nycteribiid density support the role of bat flies as vectors of bartonellae. The study provides novel information on comparative prevalence and genetic diversity of Bartonella in pteropodid bats and their ectoparasites, as well as demographic factors that affect Bartonella transmission and potentially other bat-borne pathogens.

Bacterial microbiota play an important role in the fitness of arthropods, but the bacterial microflora in the parasitic mite Dermanyssus gallinae is only partially explored; there are gaps in our understanding of the microbiota localization and in our knowledge of microbial community assembly. In this work, we have visualized, quantified the abundance, and determined the diversity of bacterial occupancy, not only across developmental stages of D. gallinae, but also in the midgut of micro-dissected female D. gallinae mites. We explored community assembly and the presence of keystone taxa, as well as predicted metabolic functions in the microbiome of the mite. The diversity of the microbiota and the complexity of co-occurrence networks decreased with the progression of the life cycle. However, several bacterial taxa were present in all samples examined, indicating a core symbiotic consortium of bacteria. The relatively higher bacterial abundance in adult females, specifically in their midguts, implicates a function linked to the biology of D. gallinae mites. If such an association proves to be important, the bacterial microflora qualifies itself as an acaricidal or vaccine target against this troublesome pest.

Climatic change conditions (elevated CO2 and warming) have been known to threaten agricultural sustainability and grain yield. Soil fungi play an important role in maintaining agroecosystem functions. However, little is known about the responses of fungal community in paddy field to elevated CO2 and warming. Herein, using internal transcribed spacer (ITS) gene amplicon sequencing and co-occurrence network methods, the responses of soil fungal community to factorial combinations of elevated CO2 (550 ppm), and canopy warming (+2 °C) were explored in an open-air field experiment for 10 years. Elevated CO2 significantly increased the operational taxonomic unit (OTU) richness and Shannon diversity of fungal communities in both rice rhizosphere and bulk soils, whereas the relative abundances of Ascomycota and Basidiomycota were significantly decreased and increased under elevated CO2, respectively. Co-occurrence network analysis showed that elevated CO2, warming, and their combination increased the network complexity and negative correlation of the fungal community in rhizosphere and bulk soils, suggesting that these factors enhanced the competition of microbial species. Warming resulted in a more complex network structure by altering topological roles and increasing the numbers of key fungal nodes. Principal coordinate analysis indicated that rice growth stages rather than elevated CO2 and warming altered soil fungal communities. Specifically, the changes in diversity and network complexity were greater at the heading and ripening stages than at the tillering stage. Furthermore, elevated CO2 and warming significantly increased the relative abundances of pathotrophic fungi and reduced those of symbiotrophic fungi in both rhizosphere and bulk soils. Overall, the results indicate that long-term CO2 exposure and warming enhance the complexity and stability of soil fungal community, potentially threatening crop health and soil functions through adverse effects on fungal community functions.

Alien species can host diverse microbial communities. These associated microbiomes may be important in the invasion process and their analysis requires a holistic community-based approach. We analysed the skin and gut microbiome of Eleutherodactylus johnstonei from native range populations in St Lucia and exotic range populations in Guadeloupe, Colombia, and European greenhouses along with their respective environmental microbial reservoir through a 16S metabarcoding approach. We show that amphibian-associated and environmental microbial communities can be considered as meta-communities that interact in the assembly process. High proportions of bacteria can disperse between frogs and environment, while respective abundances are rather determined by niche effects driven by the microbial community source and spatial environmental properties. Environmental transmissions appeared to have higher relevance for skin than for gut microbiome composition and variation. We encourage further experimental studies to assess the implications of turnover in amphibian-associated microbial communities and potentially invasive microbiota in the context of invasion success and impacts. Within this novel framework of "nested invasions," (meta-)community ecology thinking can complement and widen the traditional perspective on biological invasions.

Many studies have noted differences in microbes associated with animals reared in captivity compared to their wild counterparts, but few studies have examined how microbes change when animals are reintroduced to the wild after captive rearing. As captive assurance populations and reintroduction programs increase, a better understanding of how microbial symbionts respond during animal translocations is critical. We examined changes in microbes associated with boreal toads (Anaxyrus boreas), a threatened amphibian, after reintroduction to the wild following captive rearing. Previous studies demonstrate that developmental life stage is an important factor in amphibian microbiomes. We collected 16S marker-gene sequencing datasets to investigate: (i) comparisons of the skin, mouth, and fecal bacteria of boreal toads across four developmental life stages in captivity and the wild, (ii) tadpole skin bacteria before and after reintroduction to the wild, and (iii) adult skin bacteria during reintroduction to the wild. We demonstrated that differences occur across skin, fecal, and mouth bacterial communities in captive versus wild boreal toads, and that the degree of difference depends on developmental stage. Skin bacterial communities from captive tadpoles were more similar to their wild counterparts than captive post-metamorphic individuals were to their wild counterparts. When captive-reared tadpoles were introduced to a wild site, their skin bacteria changed rapidly to resemble wild tadpoles. Similarly, the skin bacterial communities of reintroduced adult boreal toads also shifted to resemble those of wild toads. Our results indicate that a clear microbial signature of captivity in amphibians does not persist after release into natural habitat.

Denitrification in sediments is a key microbial process that removes excess fixed nitrogen, while dissimilatory nitrate reduction to ammonium (DNRA) converts nitrate to ammonium. Although microorganisms are responsible for essential nitrogen (N) cycling, it is not yet fully understood how these microbially mediated processes respond to toxic hydrophobic organic compounds (HOCs) and metals. In this study, we sampled long-term polluted sediment from the outer harbor of Oskarshamn (Baltic Sea), measured denitrification and DNRA rates, and analyzed taxonomic structure and N-cycling genes of microbial communities using metagenomics. Results showed that denitrification and DNRA rates were within the range of a national reference site and other unpolluted sites in the Baltic Sea, indicating that long-term pollution did not significantly affect these processes. Furthermore, our results indicate an adaptation to metal pollution by the N-cycling microbial community. These findings suggest that denitrification and DNRA rates are affected more by eutrophication and organic enrichment than by historic pollution of metals and organic contaminants.

The hot spring Vranjska Banja is the hottest spring on the Balkan Peninsula with a water temperature of 63-95 °C and a pH value of 7.1, in situ. According to the physicochemical analysis, Vranjska Banja hot spring belongs to the bicarbonated and sulfated hyperthermal waters. The structures of microbial community of this geothermal spring are still largely unexplored. In order to determine and monitor the diversity of microbiota of the Vranjska Banja hot spring, a comprehensive culture-independent metagenomic analysis was conducted in parallel with a culture-dependent approach for the first time. Microbial profiling using amplicon sequencing analysis revealed the presence of phylogenetically novel taxa, ranging from species to phyla. Cultivation-based methods resulted in the isolation of 17 strains belonging to the genera Anoxybacillus, Bacillus, Geobacillus, and Hydrogenophillus. Whole-genome sequencing of five representative strains was then performed. The genomic characterization and OrthoANI analysis revealed that the Vranjska Banja hot spring harbors phylogenetically novel species of the genus Anoxybacillus, proving its uniqueness. Moreover, these isolates contain stress response genes that enable them to survive in the harsh conditions of the hot springs. The results of the in silico analysis show that most of the sequenced strains have the potential to produce thermostable enzymes (proteases, lipases, amylases, phytase, chitinase, and glucanase) and various antimicrobial molecules that can be of great importance for industrial, agricultural, and biotechnological applications. Finally, this study provides a basis for further research and understanding of the metabolic potential of these microorganisms.

Staphylococcus aureus is one of the most prevalent pathogens causing bovine mastitis in the world, in part because of its ease of adaptation to various hosts and the environment. This study aimed to determine the prevalence of S. aureus in Colombian dairy farms and its relationship with the causal network of subclinical mastitis. From thirteen dairy farms enrolled, 1288 quarter milk samples (QMS) and 330 teat samples were taken from cows with positive (70.1%) and negative California Mastitis Test (CMT). In addition, 126 samples from the milking parlor environment and 40 from workers (nasal) were collected. On each dairy farm, a survey was conducted, and the milking process was monitored on the day of sampling. S. aureus was identified in 176 samples, i.e., 138 QMS, 20 from teats, 8 from the milking parlor environment, and 10 from workers' nasal swabs. Isolates identified as S. aureus underwent proteomics (clustering of mass spectrum) and molecular (tuf, coa, spa Ig, clfA, and eno genes) analysis. Regarding proteomics results, isolates were distributed into three clusters, each with members from all sources and all farms. Concerning molecular analysis, the virulence-related genes clfA and eno were identified in 41.3% and 37.8% of S. aureus isolates, respectively. We provide evidence on the circulation of S. aureus strains with limited variability among animals, humans, and the environment. The parameters with the lowest compliance in the farms which may be implicated in the transmission of S. aureus are the lack of handwashing and abnormal milk handling.

The surface water is an important habitat for freshwater microorganisms, but there is a lack of understanding of the pattern of microbial diversity and structure in stream continuums of small subtropical forest watersheds. Therefore, this study aimed to understand the variations in microbial diversity and community structure along stream orders (1-5) in the small subtropical forest catchments of the Wuyi Mountains. Using GIS software, 20 streams were chosen and classified into 5 orders. Illumina sequencing was used to analyze the dynamics of microbial communities, along with stream orders and hydro-chemical properties of stream water were also determined. Our results indicated that the bacterial and fungal richness (ACE index) was higher in low-order (1 and 2 orders) streams than in high-order (3, 4, and 5 orders) streams, with the highest value in the order 2 streams (P < 0.05). The water temperature and dissolved oxygen were positively correlated with fungal richness (P < 0.05). The bacterial rare taxa had a significant correlation with the abundance taxa (P < 0.05). The relative abundances of Bacteroidetes, Actinobacteria, and Chytridiomycota microbial phyla were significantly different among different order streams (P < 0.05). Using the neutral community model, we found that the fungal community structure was significantly shaped by hydro-chemical properties, while the bacterial community structure was largely regulated by stochastic processes. Our findings suggest that variations in microbial community structure in subtropical headwaters are largely shaped by the water temperature and dissolved oxygen.

Hot spring biofilms are stable, highly complex microbial structures. They form at dynamic redox and light gradients and are composed of microorganisms adapted to the extreme temperatures and fluctuating geochemical conditions of geothermal environments. In Croatia, a large number of poorly investigated geothermal springs host biofilm communities. Here, we investigated the microbial community composition of biofilms collected over several seasons at 12 geothermal springs and wells. We found biofilm microbial communities to be temporally stable and highly dominated by Cyanobacteria in all but one high-temperature sampling site (Bizovac well). Of the physiochemical parameters recorded, temperature had the strongest influence on biofilm microbial community composition. Besides Cyanobacteria, the biofilms were mainly inhabited by Chloroflexota, Gammaproteobacteria, and Bacteroidota. In a series of incubations with Cyanobacteria-dominated biofilms from Tuhelj spring and Chloroflexota- and Pseudomonadota-dominated biofilms from Bizovac well, we stimulated either chemoorganotrophic or chemolithotrophic community members, to determine the fraction of microorganisms dependent on organic carbon (in situ predominantly produced via photosynthesis) versus energy derived from geochemical redox gradients (here simulated by addition of thiosulfate). We found surprisingly similar levels of activity in response to all substrates in these two distinct biofilm communities, and observed microbial community composition and hot spring geochemistry to be poor predictors of microbial activity in the study systems.

Protists are essential components of soil microbial communities, mediating nutrient cycling and ecosystem functions in terrestrial ecosystems. However, their distribution patterns and driving factors, particularly, the relative importance of climate, plant and soil factors, remain largely unknown. This limits our understanding of soil protist roles in ecosystem functions and their responses to climate change. This is particularly a concern in dryland ecosystems where soil microbiomes are more important for ecosystem functions because plant diversity and growth are heavily constrained by environmental stresses. Here, we explored protist diversity and their driving factors in grassland soils on the Tibetan Plateau, which is a typical dryland region with yearly low temperatures. Soil protist diversity significantly decreased along the gradient of meadow, steppe, and desert. Soil protist diversity positively correlated with precipitation, plant biomass and soil nutrients, but these correlations were changed by grazing. Structural equation and random forest models demonstrated that precipitation dominated soil protist diversity directly and indirectly by influencing plant and soil factors. Soil protist community structure gradually shifted along meadow, steppe and desert, and was driven more by precipitation than by plant and soil factors. Soil protist community compositions were dominated by Cercozoa, Ciliophora and Chlorophyta. In particular, Ciliophora increased but Chlorophyta decreased in relative abundance along the gradient of meadow, steppe and desert. These results demonstrate that precipitation plays more important roles in driving soil protist diversity and community structure than plant and soil factors, suggesting that future precipitation change profoundly alters soil protist community and functions in dry grasslands.

Sulphur, available in the form of sulphate, is one of the essential nutrients that is required by plants. Bacteria capable of oxidizing reduced forms of sulphur to sulphate play an important role in sulphur nutrition for plants. The present study was conducted to isolate, screen, and characterize sulphur-oxidizing bacteria from different soil samples collected from mustard rhizosphere and fly ash mixed soil. A total of 33 sulphur-oxidizing bacterial isolates (HMSOB1-33) were retrieved from soil and further screened for sulphur-oxidizing ability. Maximum solubilization index (3.76), pH reduction (3.93), and sulphate production (173.61 µg/ml) were observed for the isolate HMSOB2 which on the basis of 16S rDNA sequencing was identified as Pantoea dispersa with sequence similarity 98.22%. Four other selected bacterial isolates were identified as Bacillus megaterium, Bacillus tropicus, Bacillus velezensis, and Bacillus cereus. Sulphate solubilization index (SSI) correlated positively (r = 0.91) with sulphate production; however, pH showed negative correlation (r =  - 0.82) with SSI as well as sulphate production after 120 h of incubation. These promising bacterial isolates could be further explored as bioinoculant after assessing plant growth traits.

The introduction of legumes into rotations can improve nitrogen use efficiency and crop yield; however, its microbial mechanism involved remains unclear. This study aimed to explore the temporal impact of peanut introduction on microorganisms related to nitrogen metabolism in rotation systems. In this study, the dynamics of diazotrophic communities in two crop seasons and wheat yields of two rotation systems: winter wheat - summer maize (WM) and spring peanut → winter wheat - summer maize (PWM) in the North China Plain were investigated. Our results showed that peanut introduction increased wheat yield and biomass by 11.6% (p < 0.05) and 8.9%, respectively. Lower Chao1 and Shannon indexes of the diazotrophic communities were detected in soils that sampling in June compared with those sampling in September, although no difference was found between WM and PWM. Principal co-ordinates analysis (PCoA) showed that rotation system significantly changed the diazotrophic community structures (PERMANOVA; p < 0.05). Compared with WM, the genera of Azotobacter, Skermanella, Azohydromonas, Rhodomicrobium, Azospirillum, Unclassified_f_Opitutaceae, and Unclassified_f_Rhodospirillaceae were significantly enriched (p < 0.05) in PWM. Furthermore, rotation system and sampling time significantly influenced soil properties, which significantly correlated with the top 15 genera in relative abundance. Partial least squares path modeling (PLS-PM) analysis further showed that the diazotrophic community diversity (alpha- and beta-diversity) and soil properties (pH, SOC and TN) significantly affected wheat yield. In conclusion, legume inclusion has the potential to stabilize diazotrophic community structure at the temporal scales and increase subsequent crop yield.

Microbial catabolic activity (MCA) defined as the degrading activity of microorganisms toward various organic compounds for their growth and energy is commonly used to assess soil microbial function potential. For its measure, several methods are available including multi-substrate-induced respiration (MSIR) measurement which allow to estimate functional diversity using selected carbon substrates targeting specific biochemical pathways. In this review, the techniques used to measure soil MCA are described and compared with respect to their accuracy and practical use. Particularly the efficiency of MSIR-based approaches as soil microbial function indicators was discussed by (i) showing their sensitivity to different agricultural practices including tillage, amendments, and cropping systems and (ii) by investigating their relationship with soil enzyme activities and some soil chemical properties (pH, soil organic carbon, cation exchange capacity). We highlighted the potential of these MSIR-based MCA measurements to improve microbial inoculant composition and to determine their potential effects on soil microbial functions. Finally, we have proposed ideas for improving MCA measurement notably through the use of molecular tools and stable isotope probing which can be combined with classic MSIR methods. Graphical abstract describing the interrelation between the different parts and the concepts developed in the review.