Microorganisms naturally present at environmental contaminated sites are capable of biodegrading, biotransforming, or removing contaminants in soil and groundwater through bioremediation processes. Cleanup strategies and goals for site remediation can be effectively achieved by bioremediation leveraging the capabilities of microorganisms to biotransform contaminants into lesser or non-toxic end products; however, reproducible success can be limited by inadequate design or performance monitoring. A group of biological analyses collectively termed molecular biological tools (MBTs) can be used to assess the contaminant-degrading capabilities and activities of microorganisms present in the environment and appropriately implement bioremediation approaches. While successful bioremediation has been demonstrated through previously described lab-scale studies and field-scale implementation for a variety of environmental contaminants, design and performance monitoring of bioremediation has often been limited to inferring biodegradation potential, occurrence, and pathways based on site geochemistry or lab-scale studies. Potential field-scale application of MBTs presents the opportunity to more precisely design and monitor site-specific bioremediation approaches. To promote standardization and successful implementation of bioremediation, a framework for field-scale application of MBTs within a multiple lines of evidence (MLOE) approach is presented. The framework consists of three stages: (i) “Assessment” to evaluate naturally occurring biogeochemical conditions and screen for potential applicability of bioremediation, (ii) “Design” to define a site-specific bioremediation approach and inform amendment selection, and (iii) “Performance Monitoring” to generate data to measure or infer bioremediation progress following implementation. This framework is introduced to synthesize the complexities of environmental microbiology and guide field-scale application of MBTs to assess bioremediation potential and inform site decision-making. There is growing interest in applying natural approaches to contaminated site management, such as bioremediation which may have lower energy or resource consumption as compared to mechanical or chemical remediation approaches . BioremeDuring the past 20 years, molecular biological tools (MBTs) have been increasingly utilized due to scientific advancements and decreased analytical costs, to directly assess microbiological processes at environmental sites . MBTs, sWhile prior publications have focused on the advantages of employing MBTs in concert with contaminant chemistry and geochemistry evaluations to reduce site uncertainties and better characterize subsurface microbiology , it has In this perspective, a framework is introduced to synthesize the complexities of environmental microbiology and guide field-scale application of MBTs to assess bioremediation potential and inform site decision-making. This framework consists of following a staged process and a multiple lines of evidence (MLOE) approach to meet site-specific objectives associated with contaminant biodegradation or biotransformation using field-scale data. Example application of this framework can be found in another article in this research topic issue .2) (3–), oxidized manganese (Mn3+/4+), oxidized iron (Fe3+), sulfate (SO42–), and carbon dioxide (CO2). Since utilizing different electron acceptors yield varying energies , microorganisms prefer to utilize the electron acceptor with the highest energy gain first resulting in a preferential use sequence of O2 > NO3– > Mn3+/4+ > Fe3+ > SO42– > CO2  . The ele2– > CO2 . A schemAt the time of writing this perspective, commercially available and scalable MBTs which can be more readily applied at field-scale to support site management decision-making within a MLOE approach are nucleic acid-based analyses that include qPCR, reverse transcriptase qPCR (RT-qPCR), and next generation sequencing (NGS) of the 16S rRNA gene.Dehalogenimonas spp.  which reduces benzoyl-coenzyme A  , key funnas spp. . The preRT-qPCR is a variation of qPCR, which can be used to quantify gene expression  indicative of biological activity. The occurrence of mRNA expressed from genes linked to biodegradation indicates that the associated contaminant-degrading microorganisms are metabolically active and carrying out biodegradation processes . While pNGS of the 16S rRNA gene can be used to identify microorganisms in a sample and assess the composition of the microbial community. The 16S rRNA gene is evaluated to identify and assess prokaryotes (bacteria and archaea) at contaminated sites. NGS can most relevantly be used when gene targets linked to contaminant biodegradation  are not known, and to further screen trends in community composition to identify genera or species with potential contaminant-degrading capabilities . These dIn addition to these nucleic acid-based MBTs, there are other approaches that can be leveraged to measure or infer contaminant biodegradation, such as proteomics, metabolomics, and isotope-based methods. While proteomics and metabolomics measuring enzymes or intermediates are less often currently used at contaminated sites, expanded use of these MBTs in the future is anticipated. Further, isotope-based methods, such as compound specific isotope analysis (CSIA), are currently considerably used at contaminated sites and can be considered where applicable .The framework structure  proposedAt the onset of applying this framework, historical site data should be reviewed to understand the nature, extent, trends of contamination and associated site risk-drivers . HydrogeThe objective of the assessment stage is to identify potential biodegradation or biotransformation processes to inform the suitability of bioremediation. The assessment stage is where field-scale MBT data may be first generated to develop or refine the site biogeochemical CSM. While the assessment stage within this framework is not to be confused with project site characterization and is applicable across the project lifecycle where bioremediation may be considered , the generation of MBT data should be done intentionally to support answering site-specific questions or achieving site-specific objectives .A sampling plan should be developed and implemented in alignment with SSBOs, CSM, and regulatory requirements. Samples should be collected at target locations across the area(s) of interest related to contaminant(s) concentration and geochemical gradients and include unimpacted background location(s) . The goaSubsequently generated contaminant, geochemical, and MBT data can be interpreted through spatial and/or temporal comparative analysis to support determination of bioremediation potential. MBT data interpretation should be aligned with previously developed MLOE approaches and/or MNA guidance, and should consist of assessing contaminant trends for direct or indirect evidence of biodegradation, geochemical conditions to identify biodegradation mechanisms and potential electron acceptor/donor limitations of biological activity, presence and abundance of contaminant-degrading microorganisms or genes, and the inferred extent of intrinsic biodegradation . This apIf the results of Stage 1 suggest SSBOs have not been met nor determined infeasible to meet, the framework proceeds into the design stage (Stage 2). When a natural bioremediation strategy is posited to accomplish site remediation objectives, the generated contaminant, geochemical, and MBT datasets from Stage 1 should provide sufficient, conclusive evidence to support occurrence of intrinsic bioremediation across the area of interest. In addition to demonstrating natural bioremediation, the Stage 1 MLOE datasets should guide the monitoring program design including selection of sample locations  and sampling parameters to assess occurrence of natural bioremediation as a mechanism of natural attenuation.Dehalococcoides spp. and stimulated complete reductive dechlorination of tetrachloroethene (PCE), trichloroethene (TCE) and daughter products cis-1,2-dichloroethene and vinyl chloride to non-toxic end product ethene , lactate, formate], electron acceptor , nutrients , pH, temperature}, and/or microbial cultures . Additionally, Stage 1 data should be used to support the estimation of amendment dosing, location and frequency based on observed biogeochemical processes and stoichiometry. Field-scale pilot testing is recommended to be performed to refine amendment dosing and frequency, and field-scale feasibility and effectiveness prior to full-scale implementation. Geochemical parameters are utilized to monitor the biogeochemical effects of the approach while qPCR or other MBTs monitor the effects on the microbial population in response to biostimulation or bioaugmentation. This approach was employed in a pilot and full-scale remedial application to demonstrate organic carbon amendment addition promoted growth of t ethene .Performance monitoring (Stage 3) follows design and initial implementation of the natural or enhanced bioremediation strategy. While contaminant concentrations ultimately must show progress toward the remedial goals, sampling and analysis of select geochemical parameters and MBTs during remedy performance monitoring can improve understanding of remedy progress. MBT application may consist of qPCR analyses to assess whether contaminant-degrading microorganisms are sufficiently abundant to sustain desired biodegradation. Temporal and spatial trends for qPCR results can be particularly helpful to demonstrate positive, negligible, or negative effects on microbial processes to refine the remedial strategy across the treatment area, and in some cases serve as a leading indicator of remedy performance. Contaminant concentrations can, in some cases, be a lagging indicator of enhanced bioremediation due to various fate and transport mechanisms which may act as confounding factors impeding the ability to distinguish occurrence or enhancement of contaminant biodegradation. For example, as the rates of contaminant biodegradation are increased due to successful enhanced bioremediation, the rate of dissolution of residual non-aqueous phase liquids (NAPL) may also increase to maintain multi-phase equilibrium , 2009. AbssA gene (involved in the first step of anaerobic degradation of toluene and xylene; Data generated in Stage 3 should be continually reviewed to assess remedy performance monitoring. Data interpretation should be done as described in section “Stage 1: Assessment,” consisting of comparative analysis across contaminant and geochemical concentration gradients, within a MLOE approach, and compared to SSBOs, CSM, and regulatory requirements. In a study where sulfate was introduced to the subsurface, an integrated performance monitoring program of contaminants, geochemical parameters, and MBTs demonstrated that sulfate injection increased the rates of biodegradation of BTEX . As sulfAt the conclusion of the framework, whether having progressed through all three stages or solely Stage 1, a biogeochemical CSM should have been developed or refined to support subsequent site decision-making and strategy. Microbiology plays an important role in contaminant fate and attenuation, but is often times unmeasured, inferred, or not considered at contaminated sites. Applying this framework will aid in improving knowledge of microbial processes and broader contaminant fate at contaminated sites. Additionally, data-driven decisions based on this framework should improve the effectiveness of natural or enhanced bioremediation to meet site goals and achieve or progress toward site closure, as well as avoid the application of bioremediation where it may be infeasible.Bioremediation methods have been demonstrated to be favorable strategies for contaminated site management to naturally biodegrade, biotransform or remove contaminants in soil and groundwater. As discussed in this perspective, the increasing application of MBTs to directly measure microbiological processes at environmental sites has the potential to reduce uncertainty related to site-specific biogeochemical processes. However, field-scale MBT application, implementation strategies, and interpretation can be inconsistent across varying sectors, institutions, or individuals potentially posing barriers to increased uptake and acceptance. For this reason, the field-focused framework described herein was developed to synthesize complexities of environmental microbiology and assess bioremediation potential at the field-scale during various stages of planning and execution.Application of this framework should be considered for use at contaminated sites implementing bioremediation; further description of application can be found in another article in this research topic issue . It shouThe original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.TK and AM contributed to the conception and design of this perspective. SS and YW refined and sharpened conceptual layout and messaging of content. TK, SS, and AM wrote sections of the manuscript. All authors contributed to the manuscript review, read, and approved the submitted manuscript.


We find that the main glacier lobe underwent profound retreat after 19.7 ± 0.7 ka. This recessional trend led to the individualization of the Cerro Riñón glacier by ~ 16.3 ka, which underwent minor readvances at 15.9 ± 0.5 ka during Heinrich Stadial 1, during the Antarctic Cold Reversal with successive maxima at 13.5 ± 0.4, 13.1 ± 0.4, and 13.1 ± 0.5 ka, and a minor culmination at 12.5 ± 0.4 ka during Younger Dryas time. We conclude that fluctuations of Patagonian glaciers during T1 were controlled primarily by climate anomalies brought by shifts in the Southern Westerly Winds (SWW) locus. We posit that the global covariation of mountain glaciers during T1 was linked to variations in atmospheric CO2 (atmCO2) promoted by the interplay of the SWW-Southern Ocean system at millennial timescales.The Last Glacial Termination (T1) featured major changes in global circulation systems that led to a shift from glacial to interglacial climate. While polar ice cores attest to an antiphased thermal pattern at millennial timescales, recent well-dated moraine records from both hemispheres suggest in-phase fluctuations in glaciers through T1, which is inconsistent with the bipolar see-saw paradigm. Here, we present a glacier chronology based on 30 new  T1 featured the collapse of continental-scale ice sheets, a persistent rise in both sea level and the concentration of atmospheric greenhouse gases, along with a millennial-scale sequence of climate changes that culminated with the onset of the current interglacial. Ice core data reveal a step-like pattern of increasing atmospheric temperatures featuring a synchronous but antiphased trend at millennial timescale between the polar hemispheres3. Recently developed precise mountain glacier chronologies in the mid-to-high latitudes of both hemispheres7  represents the largest, most abrupt climate change of the last glacial-interglacial cycle8 proposed that changes in the Southern Westerly Winds (SWW) are the critical missing link relating insolation, atmospheric greenhouse gas concentrations, atmospheric and ocean circulation, and glacier response at millennial timescale during glacial maxima and terminations. By way of interaction with the Southern Ocean (SO), the SWW establish a coupled system that drives global ocean circulation, the atmospheric concentration of greenhouse gases, and high-latitude ocean productivity by enhancing upwelling of CO2-enriched and high-nutrient deep waters9. The efficacy of the SWW-SO coupled system is dependent upon the wind stress imparted by the SWW on the surface of the SO south of the Drake Passage (> 55° S)10. Hence, deciphering the geographical position and strength of the locus of the SWW are crucial for assessing global climate change during ice age terminations.Recently, Denton et al.11. This region-specific response to incident atmospheric flow allows reconstructing past SWW behavior based on hydrologic balance variations preserved in stratigraphic and geomorphologic records from the Chilean Lake District and Chilotan archipelago (40–44° S). When analyzed in conjunction with similar records from southern Patagonia (50°-54°S), latitudinal shifts and intensity variations in the SWW at millennial-scale during the Last Glacial Maximum  and T113 can be identified. However, few studies in northern Patagonia have examined in any detail glacier fluctuations following the onset of T115, limiting our understanding about the response of middle latitude austral glaciers and paleoclimate patterns and processes at continental, hemispheric, and global scales.The SWW are the sole driver of precipitation to the Pacific and Andean divide sectors of northern Patagonia (40°–44° S)16, we present a glacial geomorphologic map and a 10Be geochronology of the inner Lago Palena/General Vintter (LPGV) basin, centered at ~ 43.9° S; ~ 71.5° W in northern Patagonia, to examine the timing and structure of glacier fluctuations during T1. This basin was covered by an eastward-flowing outlet lobe of the Patagonian Ice Sheet that was fed through several coalescing valleys during the LGM . The Cerro Riñón valley  is carved into the North Patagonian batholith18, which was the source of the abundant glacially-transported granitoid boulders found on the local landscape.Mountain glaciers are sensitive to and provide a direct physical link to changing atmospheric conditions. For land-terminating glaciers in particular, moraines distal to present ice limits are unambiguous recorders that past climate changed. Thus, the anatomy of glacier fluctuations during T1 in the mid-latitude Andes not only offers empirical constraints on the interhemispheric synchrony of mountain glacier behavior, but also a means to examine the evolution of the SWW and associated paleoclimates. Building on prior mapping19. Our geomorphological map allows identification of at least six well-preserved, closely spaced arcuate moraine ridges, the innermost of which we name PV6 . A single 10Be sample from a perched erratic boulder resting over polished bedrock outboard of the CR1 moraines, and ~ 30 m above the modern lake surface, affords an age of 16.3 ± 0.4 ka. We interpret this date as a minimum-limiting age for local ice evacuation. The CR1 moraines lie ~ 300 m south from the lake shore and comprise a ~ 500 m long and ~ 10 m high main ridge connected to several minor ridges  and sharper appearance  and prominent (~ 30 m high) ice-marginal feature of the area. We obtained seven 10Be samples that range in age from 13.5 ± 0.3 to 12.9 ± 0.3 ka, with a mean of 13.1 ± 0.4 ka. Approximately 200 m upstream from CR3, separated by an outwash plain, a group of several discontinuous ridges covered by dense vegetation form the CR4 moraine   constitute the basis of a moraine chronology for the LPGV basin that documents in detail the sequence of glacier/paleoclimatic events during T1 in northern Patagonia. Our data indicate that the retreat of the LPGV glacier lobe from the PV6 moraine at 19.7 ± 0.7 ka likely initiated the present lake, and the ice front did not subsequently re-advance out of its current basin. Considering the dispersion of the PV6 ages, we interpret this date as a maximum-limiting age for presumed large-scale glacier withdrawal during T1. The ice front then retreated more than 40% of its LGM length, prompting the detachment of the Cerro Riñón glacier shortly after 16.3 ± 0.4 ka. After this event, a moraine-building event of this tributary glacier culminated with the deposition of the CR1 moraine at 15.9 ± 0.5 ka. Subsequent advances or standstills deposited in quick succession within the error margin of the dating the CR2 to CR4 moraines between 13.5 ± 0.4 and 13.1 ± 0.4 ka. Cerro Riñón glacier then underwent net recession, only interrupted by a stillstand that constructed CR5 at 12.5 ± 0.4 ka. No ice marginal features are evident up valley, suggesting profound glacier retreat to the headwalls after ~ 12.5 ka. The chronology for the Cerro Riñón glacier informs on the timing and structure of glacier fluctuations throughout the entirety of the T1 chron within a single basin in northern Patagonia, and constrains four moraine deposition phases of similar magnitude  that culminated at ~ 15.9 ka during Heinrich Stadial 1 (HS1: ~ 17.8–14.7 ka) and between ~ 13.5–13.1 ka within the Antarctic Cold Reversal (ACR: ~ 14.7–12.7 ka), followed by an additional advance or stillstand of minor extent  at ~ 12.5 ka that is coeval with the Younger Dryas (YD: ~ 12.6–11.5 ka).Glacial geologic mapping and thirty new 10Be dated Cerro Pintado moraine at Río Guanaco (~ 50°S)24 and the 14C-constrained Lago Pinto moraine at Última Esperanza (~ 50°S)25. Subsequent to HS1, glacier activity in the Cerro Riñón valley represents the northernmost direct evidence for glacier advances during the ACR and gradual retreat during the YD in Patagonia, expanding the known geographical footprint of these glacier/paleoclimate events from 47.5o S to 43.9o S29. The recent interpretation of an ACR maximum based on geomorphic analysis and lake sedimentary record of former Rosselot glacier30, ~ 45 km directly west of Cerro Riñón, is consistent with our results. Considering that Patagonian ice-marginal features formed during the early phases of T1 are often closely spaced, these findings indicate that equilibrium line altitude changes (ELA) at ~ 15.9 ka and during the ACR (CR1-CR4) were similar in magnitude and were followed by a net ELA rise during the YD (CR5), accounting for modest glacier advances or standstills well within ACR limits. At similar latitude, but ~ 9000 km west of LPGV, several moraine records in New Zealand reflect synchronous glacier behavior: massive recession from the LGM limits was punctuated by deposition of the Prospect Hill moraines at ~ 15.9 ka in the Rakaia valley31, which was followed by several ACR advances, and then subsequent YD recession was interrupted by minor stillstands in multiple valleys of the Southern Alps36 . These changes suggest successive incremental increases for SWW influence in northern Patagonia. The earliest of these increases on the wind belt influence lasted until ~ 15.9 ka, as the deglacial warming trend resumed and crossed a critical threshold that favored the diversification and densification of other thermophilous rainforest trees and vines at the expense of Fitzroya/Pilgerodendron. The youngest increase in conifers (Podocarpus nubigena) during T1 took place between ~ 14.7–12.6 ka, and was followed by enhanced fire activity, a lake-level fall, and decline in conifers after ~ 12.6 ka, which suggest a decrease in precipitation related to a southward shift of the SWW. Wind-driven hydroclimate changes toward cold/wet conditions closely track glacier advances in the LPGV area. We therefore conclude that our record shows a coherent cryospheric response to significant SWW-modulated climate fluctuations during T1. In addition, we note that the reservoir-age corrected paleoclimate records from marine core ODP123341, collected offshore from northwestern Patagonia  changes recorded at the beginning and end of T1, between ~ 18.1–16.3 ka and ~ 13–11.7 ka in the WAIS ice core43, enhanced ocean ventilation inferred from a rise in opal flux recorded in the SO9, and reduced δ13C composition of atmCO2 preserved in the EPICA Dome C (EDC) and the Talos Dome (TALDICE) ice cores from Antarctica44  and southern  hemispheres. From our interhemispheric comparison we interpret synchrony of mountain glaciers driven by a global climate signal during T1, challenging the expected antiphase behavior predicted from the bipolar seesaw paradigm. This finding favors an atmospheric mechanism for generating and globally propagating millennial-scale climate variability during T1. We conclude that high-to-middle latitude mountain glaciers fluctuated in phase during T1 partly in response to summer warming due to atmCOwww.saf.cl), satellite imagery (Sentinel 2—https://scihub.copernicus.eu/) and digital elevation models . The preliminary map was checked during two field campaigns .Detailed geomorphological mapping of the moraine limits in Lago Palena/General Vintter basin was conducted based on aerial photographs , in addition to one sample from a perched boulder (LV17-42) located immediately outboard of the outermost moraine limit, and 4 boulder samples from the innermost moraine ridge along the eastern shore of Lago Palena/General Vintter . We aimed for large boulders to avoid potential effects of post-depositional movements or posterior exhumation. Samples were taken from the upper ~ 4 cm of the boulder surfaces using a drill and explosive charges, avoiding areas exhibiting clear signs of erosion, such as spalling or flaking. Elevation and geographic coordinates of each rock sample were recorded with a handheld GPS unit (WGS84). We measured topographic shielding using a handheld compass and a clinometer. Moraine ages are interpreted as representing the culmination of moraine construction, and, thus, cold episodes, whereas the perched-boulder age provides a minimum age for ice retreat from the sampling site.We collected 26 boulder samples for 60. 10Be/9Be ratios were measured at Lawrence Livermore National Laboratory Center for Accelerator Mass Spectrometry. Ratios were measured relative to the standard 07KNSTD with a 10Be/9Be ratio of 2.85 × 10–12 . Analytical raw data are available in the supplementary material 60, the average analytical uncertainty is ~ 2%, with almost half the analyses between 1.7% and 2.0%.Initial crushing and sieving of the rock samples was carried out at the Pontificia Universidad Católica de Chile and subsequent quartz and beryllium extraction at the Cosmogenic Nuclide Laboratory at Lamont-Doherty Earth Observatory following the protocol outlined in10Be ages were calculated based on methods incorporated in the online exposure age calculator (v.3—https://hess.ess.washington.edu/math/v3/v3_age_in.html)62, considering the time-dependent Lal/Stone (Lm)22 scaling schemes and the regional Patagonian production rate (~ 50o S)20, assuming zero erosion and a rock density of 2.65 g/cm3. We discuss ages based on Lm scaling scheme because it produces the ages that best fit with limiting radiocarbon data from the production-rate calibration site at Lago Argentino (~ 50° S)20. In addition, most of the Patagonia 10Be glacial chronologies were reported according to the same scaling scheme. We show in Table 10Be ages are statistically identical  using other scaling methods. In the text, we report individual 10Be ages with 1σ analytical uncertainty and the standard error of the mean (SEM). Raw geographic and 10Be analytical data can be found in the Supplementary material Table 20. Similarly, we recalculated available glacier 10Be chronologies from New Zealand8 by using the online exposure age calculator (v.3)62 incorporating a local production rate (~ 43.6° S)63. Recalculated 10Be ages are reported with the associated uncertainties (1σ), including a 2% propagation of the production rate error63 (See Supplementary material Tables 8 used exposure age calculator v2.262 based on63 and reported cosmogenic ages calibrated to the year A.D. 1950. Therefore, our recalculated 10Be ages differ from those in Denton et al.8 by 1–2%, which does not alter our interpretations.Supplementary Information.


Botryodiplodia theobromae, Fusarium moniliforme, and Alternaria alternate) and mold fungi  to develop new chemicals to protect bamboo against stain fungi and molds. The inhibitory activity of the composite pyraclostrobin and IPBC with different ratios was evaluated. Water-based formulations of the fungi were used to treat the bamboo, and the mold and stain resistance of the bamboo was investigated at different chemical retention rates. The results showed that the antifungal activity of pyraclostrobin was significantly higher than that of azoxystrobin and kresoxim-methyl. Different degrees of inhibitory activities against the stain and mold fungi were observed, and the inhibitory activity was higher against stain fungi than against molds. The three stain fungi were completely inhibited at a 7:3 ratio of pyraclostrobin to IPBC and 0.1% concentration. As the ratio increased, the inhibitory effect against mixed mold strains improved. The control efficacy of the pyraclostrobin formulations Str-1 and Str-2 at 0.1% concentration was 100% against Alternaria alternate and 70.8% against Fusarium moniliforme. The control efficacy of the composite formulations SI-1 and SI-2 at 0.1% concentration was 100% against all three stain fungi and greater than 91.8% against the mixed mold strains. This study provides new insights into the utilization of pyraclostrobin and its composite formulations as new bamboo antifungal agents.Bamboo is rich in starch and sugars and can be infected by mold and stain fungi, degrading its performance, shortening its service life, and reducing its utilization value. It is crucial to investigate how to protect bamboo against mold and stain fungi. The zone of inhibition test was used to evaluate the antifungal activity of azoxystrobin, kresoxim-methyl, pyraclostrobin and 3-iodo-2-propynyl-butylcarbamate (IPBC) against stain fungi ( Trichoderma, Aspergillus, Penicillium, Fusarium, Alternaria, and Globisporium [Bamboo is a renewable and rapidly growing lignocellulosic plant . Due to isporium . These b2), silica dioxide (SiO2), silver (Ag) and zinc oxide (ZnO) nanoparticles, and other inorganic antifungal agents, have been used to modify bamboo and improve its mold resistance to varying degrees [Botryodiplodia theobromae and 100% against mixed mold strains at a concentration of 0.1%, i.e., the active ingredient retention of the treated bamboo was 0.39 g/m2 [In recent decades, bamboo has been protected against molds and stain fungi by influencing the conditions affecting the survival of fungi or killing them by using chemicals. For example, Moso bamboo was treated with aqueous solutions with different pH values to reduce the hemicellulose content, create conditions unsuitable for mold growth, and reduce the nutrients in the bamboo to inhibit mold growth . Bamboo  degrees ,14,15,16 degrees ,18,19. I.39 g/m2 . Unfortu.39 g/m2 . It is aStrobilurin fungicides are derived from the natural antibiotic strobilurin A with fungicidal activity, which is isolated from basidiomycetes . Since tThus, this study investigates the inhibitory activity of azoxystrobin, kresoxim-methyl, and pyraclostrobin, against bamboo stain fungi and molds using an antifungal activity test to select candidate agents and their appropriate concentrations. Several water-based agents are prepared using the candidate agents or their mixtures to treat bamboo, and the stain and mold resistance of the bamboo is evaluated. This study provides critical information for developing organic strobilurin anti-mold agents for bamboo.Azoxystrobin (purity ≥ 97%), kresoxim-methyl (purity ≥ 97%), and pyraclostrobin (purity ≥ 98%) were purchased from Wenzhou Lujia Import and Export Trade Co., Ltd., Wenzhou, China. IPBC (purity ≥ 99%) was obtained from Shanghai Huilong Chemical Co., Ltd., Shanghai, China. Agricultural emulsifiers (No. 601 and No. 1601) and castor oil polyoxyethylene ether (EL-90) were purchased from Jiangsu Haian Petrochemical Co., Ltd., Nantong, China. The other reagents are commercially available and are analytical grade; they were purchased from Sinopharm Chemical Reagent Co., Ltd., Beijing, China.Phyllostachys edulis) was purchased in Hangzhou, Zhejiang Province. The bamboo was cut into specimens with dimensions of 50 mm × 20 mm × 5 mm (L × W × H) with no mildew, knots, or other defects. The moisture content of the 5 mm specimen was 8–12%. The air-dry density and moisture content of the samples were 0.71 ± 0.04 g/cm3 and 7.3 ± 0.2%, respectively.A 4 years old bamboo plant (The stain and mold fungi isolated from bamboo were Botryodiplodia theobromae (abbreviated to Bt), Fusarium moniliforme (Fm), Alternaria alternate (Aa), Aspergillus niger (An), Penicillium citrinum (Pc) and Trichoderma viride (Tv).The zone of inhibition test was used for screening the chemicals and their concentrations. The 6 fungal strains were cultured on a PDA substrate in Petri dishes (standard 90 mm) for 7 d at 28 °C and 85% relative humidity. We put 20 mL of distilled water into a 100 mL triangular flask and added several glass balls. The flasks were plugged with cotton plugs and autoclaved for 30 min. An inoculation needle was used to remove fragments of mycelium from the edge of the fungal colonies. They were placed into cold, sterile water in a triangular bottle, which was shaken to disperse the material and obtain the fungal suspension. Each chemical was dissolved in an aqueous ethanol solution (50%) to obtain chemical solutions with mass concentrations of 0.05%, 0.1%, and 0.2%. The fungal suspension was placed on the PDA medium, and 4 Oxford cups were placed in the Petri dishes. We used a pipette to drip 0.25 mL of the chemical solutions of various concentrations and put the Petri dishes in the incubator for culture. The test was conducted on an ultra-clean workbench; a 50% ethanol solvent served as the blank control, and the IPBC solution was a positive control. The diameter of each inhibition zone was measured by vernier caliper after 2 weeks of cultivation. Each group of tests was repeated three times, and the arithmetic mean of the test value was used.B. theobromae, F. moniliforme, and A. alternate and the mixed mold fungi to determine the optimum ratio of pyraclostrobin and IPBC.We combined 0.1 g of pyraclostrobin and 99.9 g of the 50% ethanol solution in a 200 mL conical flask under magnetic stirring for 5 min to obtain a pyraclostrobin solution with 0.1% concentration. We prepared a 0.1% IPBC solution in the same way. We used a pipette to obtain 9 mL, 8 mL, 7 mL, 6 mL, 5 mL, 4 mL, 3 mL, 2 mL, and 1 mL of the 0.1% pyraclostrobin solution and mixed it with 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, and 9 mL of the 0.1% IPBC solution, respectively. Thus, we obtained nine mixed agents of pyraclostrobin and IPBC with a concentration of 0.1%. Their ratios were 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 1:9, respectively. The zone of inhibition method was used to test the inhibitory activity of the mixed agents against A mixture of 5.00 g pyraclostrobin and 20.00 g ethyl acetate was added to a 200 mL conical flask. The mixture was stirred to dissolve at 25 °C for 5 min. Then, 15.00 g of agricultural emulsifier No. 601 and 22.50 g of distilled water were dropwise added to the conical flask. The 8% pyraclostrobin formulation Str-1 was obtained after stirring. The Str-2 pyraclostrobin formulation was prepared similarly, except that the solvent was N,N-dimethylformamide, and the emulsifier was agricultural emulsifier No. 1601.The method in reference  was usedR is the active ingredient retention on the bamboo surface, g/m2; m1 is the sample weight before immersion, g; m2 is the sample weight after immersion, g; c is the mass fraction of active ingredients in the chemical solution; A is the surface area of the treated specimen, mm2.According to the results of the inhibition zone test, each formulation was diluted with water to obtain mass fraction concentrations of 0.1% and 0.2%. Next, the bamboo specimens were air-dried to a moisture content of less than 12%, weighed, and marked as m1. The specimens were immersed into the chemical solutions with different concentrations and water (control) at −0.09 MPa for 30 min and were maintained at normal pressure for 30 min. The specimens were removed from the solution, the surface was wiped clean, and they were weighed and marked as m2. We calculated the amount of retained chemicals in the bamboo specimen. The specimen was air-dried to a moisture content of 8–12%. Each experiment was replicated 6 times. The amount of active ingredient retained in the bamboo was calculated by Equation (1).The mold and stain resistance of the treated bamboo samples was evaluated according to the Chinese Standard GB/T 18261-2013 . BrieflyE) of the chemicals against mold infection on the bamboo samples was obtained from Equation (2).E is the control efficacy, %; D1 is the average infection value of the treated samples; D0 is the mean infection value of the untreated samples.The control efficacy  was used as a blank control; the results showed that it did not affect the tested strains. IPBC is a highly active anti-mold agent that was used as a positive control. The results of the inhibition zone test are listed in B. theobromae and A. alternate at various concentrations but exhibited inhibitory activity against F. moniliforme at concentrations of 0.1% and 0.2%. The diameters of the inhibition zones were 10.0 mm and 22.2 mm, respectively. Additionally, there was no inhibitory activity of kresoxim-methyl against B. theobromae, although it showed inhibitory activity against F. moniliforme and A. alternate. The diameters of the inhibition zones were 12.1–19.8 mm and 25.0–29.6 mm, respectively. In contrast, pyraclostrobin had significant inhibitory activity against all three stain fungi, and the diameter of the inhibition zone increased with an increase in the chemical concentration. The inhibitory activity against A. alternate was higher than that against B. theobromae and F. moniliforme. For example, the diameters of the inhibition zones of pyraclostrobin against B. theobromae, F. moniliforme, and A. alternate at 0.1% concentration were 20.3 mm, 20.1 mm, and 35.9 mm, respectively. IPBC is a highly effective mildew inhibitor with outstanding inhibition activity. The diameter of the inhibition zone of IPBC against the three stain fungi was larger than 45 mm at the three concentrations.The data suggested that the three chemicals had moderate to strong inhibitory activity against stain fungi . PyracloT. viride, it exhibited inhibitory activity against A. niger and P. citrinum at three concentrations. The diameters of the inhibition zones were 7.8–13.5 mm and 12.4–19.6 mm, respectively. The diameter of the inhibition zone of pyraclostrobin against P. citrinum was 21.7–26.2 mm, and those against A. niger and T. viride were 14.3–16.0 mm and 15.7–17.9 mm, respectively, at the three concentrations. Thus, pyraclostrobin exhibited higher activity against P. citrinum than against A. niger and T. viride. The inhibitory activity of IPBC against the three molds was high; the inhibition zones against A. niger and P. citrinum were greater than 49 mm at 0.1% and 0.2% concentrations, significantly higher than those of the candidate agents.In summary, pyraclostrobin exhibited higher inhibitory activity against all stain fungi and molds than the other agents. In general, its activity was slightly higher against stain fungi than against molds; thus, its ability to inhibit stain and mold fungi should be further investigated. Pyraclostrobin inhibits the respiration of mitochondria in the cells of fungi and prevents the synthesis of energy in cells, inhibiting the germination of pathogenic fungi spores ,26. IPBCThe results showed that 50% ethanol used as the blank control was inactive against the tested strains. In contrast, IPBC completely inhibited all molds and stain fungi at the concentration of 0.1% (the diameter of the inhibition zone was close to 49 mm). The concentration of the active ingredients in the chemical solution was set to 0.1%, and activity screening tests were conducted with different ratios of pyraclostrobin and IPBC to determine the optimum ratio to inhibit mold and stain fungi and minimize the amount of IPBC. The fungicidal activity against a mixture of three mold strains was tested. The results are shown in A. alternate and F. moniliforme were more than 49 mm and 46.3 mm, respectively, higher than the diameters of the 0.2% pyraclostrobin or 0.05% IPBC solutions. The three stain fungi were completely inhibited at a ratio of 7:3, whereas the inhibitory effect on the mixed mold strains was slightly weaker, with a diameter of the inhibition zone of 37.3 mm. As the ratio increased, the inhibitory effect against the mixed mold strains increased, but they were not completely inhibited. Therefore, the concentration of the active ingredients can be increased to achieve complete inhibition of molds in bamboo. According to the results of this experiment, two ratios (7:3 and 4:6) of pyraclostrobin and IPBC were selected to treat the bamboo.The results showed that the combination of pyraclostrobin and IPBC exhibited a good inhibitory effect against the tested strains. At a ratio of 9:1, the diameters of the inhibition zones against The test results of the stain and mold resistance of bamboo treated with two pyraclostrobin formulations (Str-1 and Str-2) and two composite formulations with different ratios (SI-1 and SI-2) are listed in A. alternate and 70.8% against F. moniliforme at a concentration of 0.1%, i.e., the active ingredient retention of the treated bamboo was 0.36 g/m2. The chemical retention of the treated bamboo increased with an increase in the concentration of the active ingredient. The formulations Str-1 and Str-2 did not inhibit B. theobromae at a retention of 0.65 g/m2 (0.2% concentration). However, the surfaces of the samples treated with formulations SI-1 and SI-2 at the concentration of 0.1% (retention of 0.40 g/m2) had no mycelia and mildew spots Pyraclostrobin had higher antifungal activity than azoxystrobin and kresoxim-methyl. It exhibited different degrees of inhibitory activity against all stain fungi and molds in this test. In general, its activity was slightly higher against stain fungi than against mold fungi.(2)The combination of pyraclostrobin and IPBC had a good synergistic effect. It completely inhibited the three stain fungi at a concentration of 0.1% and a ratio of pyraclostrobin to IPBC of 7:3. The diameter of the inhibition zone was 37.3 mm in the test with the mixed mold strains. (3)The mold and stain resistance of bamboo treated with pyraclostrobin formulations Str-1 and Str-2 were the same, and the control efficacy increased with an increase in the concentration of the active ingredient. The composite preparations SI-1 and SI-2 had 100% control efficacy at concentrations of 0.1% against the three stain fungi and 91.8% and 100% control efficacy, respectively, against the mold strains.This study explored the antifungal activity of strobilurin fungicides against bamboo stain fungi and molds using the zone of inhibition test and mold resistance tests. The following conclusions were drawn:


Background: Diesel exhaust particles (DEPs) are leading to a general increase in atopic diseases worldwide. However, it is still unknown whether DEPs induce systemic B-cell IgE class switching in secondary lymphoid organs or locally in the lungs in inducible bronchus-associated lymphoid tissue (iBALT). The aim of this work was to identify the exact site of DEP-mediated B-cell IgE class switching and pro-allergic antibody production. Methods: We immunized BALB/c mice with different OVA doses (0.3 and 30 µg) intranasally in the presence and absence of two types of DEPs, SRM1650B and SRM2786. We used low (30 µg) and high (150 µg) DEP doses. Results: Only a high DEP dose induced IgE production, regardless of the particle type. Local IgE class switching was stimulated upon treatment with both types of particles with both low and high OVA doses. Despite the similar ability of the two standard DEPs to stimulate IgE production, their ability to induce iBALT formation and growth was markedly different upon co-administration with low OVA doses. Conclusions: DEP-induced local IgE class switching takes place in preexisting iBALTs independent of de novo iBALT formation, at least in the case of SRM1650B co-administered with low OVA doses.  One of the main phenomena hypothesized as explaining the increase in the prevalence of different IgE-mediated pathologies such as asthma, allergic rhinitis, and atopic dermatitis is air pollution . The maiRecently, it was shown that the intratracheal instillation of monodisperse alum or silica particles caused iBALT formation and local IgE production in C57BL/6 mice . AlthougThe physical properties of DEPs vary significantly depending on their specific sources as well as the atmospheric conditions and the time they spend in the air ,22. For In our previous work, we found that low (but not high) antigen doses induced rapid local B-cell IgE class switching, accompanied by minimal IgG1 production in fat-associated lymphoid clusters rather than regional lymph nodes . HoweverThe aim of this work was to clarify (a) whether DEPs induced local (in lung tissue) or systemic (in lymph nodes) IgE class switching and IgE production when introduced with either low or high antigen doses and (b) whether local IgE class switching was accompanied by iBALT induction. We also compared the effects of two types of DEPs, SRM1650B (DEP1) and SRM2786 (DEP2), on the local and systemic immune responses.Female BALB/c mice, 5–6 weeks old, were purchased from the Scientific Center of Biomedical Technologies . The mice were housed in SPF conditions for two weeks before the experiments. During all the experimental procedures, the mice were maintained under a 12 h light/dark cycle at room temperature (20–23 °C) at 40–60% humidity in a special room in a vivarium. The mice were housed in plastic cages (10–12 mice per cage) with wood shavings as a bedding material. They were fed ad libitum with granular feed. All the animal experiments were performed according to the IACUUC protocol number 350, approved by the local committee of IBCh RAS on 21 June 2020.Before the experiments, the mice were randomly divided into experimental groups in such a manner that in each group, the average weight of the animals  did not significantly differ from the weights in the other groups and was 20–22 g. Only mice without any signs of illness that weighed more than 19 g were included in the analysis. If the animals showed signs of illness or significant depletion of the organism under the influence of the input components , they were to be withdrawn from the experiment and analysis. However, this was not observed in our work. All the mice used in the experiments had no previous contact with ovalbumin (OVA) or DEPs.n = 11–12 per group) were immunized for 8 consecutive weeks. They received saline as a control or ovalbumin  at low (0.3 µg) or high (30 µg) doses, alone or with the diesel exhaust particles (DEPs) SRM1650B (DEP1) or SRM2786 (DEP2)  3 times a week for the first 3 weeks and 2 times a week for the remaining 6 weeks. Low (30 µg/mouse) or high (150 µg/mouse) doses of DEPs were used. After immunization with different OVA and DEP doses, some mice  were challenged with 250 µg of OVA 3 times a week for 2 weeks to evaluate the intensity of allergic lung inflammation in the different groups.The overall study design is shown in The amount of DEPs administered was established according to previously published doses ,23. We ag to obtain serum samples, which were stored at −20 °C prior to use. For the ELISA experiments, six or seven mice that matched the inclusion criteria from three independent experiments were taken for general analysis.After 8 weeks of immunization, blood was taken from the suborbital sinus to estimate the specific antibody titers . The blood samples were incubated for 20 min at 37 °C, followed by centrifugation at 600× n = 6) that match the inclusion criteria from 3 independent experiments were taken for general analysis. For H&E histology, n = 6.The next day, the mice were anesthetized with isoflurane and sacrificed by cervical dislocation after the manipulations; the lung tissue and regional lymph nodes were collected and homogenized in ExtractRNA to obtain samples for gene expression measurement. Six samples per group were used for the analysis of gene and transcript expression. For CXCL13 quantification by ELISA, lungs were homogenized in a phosphate saline buffer (PBS) containing 1% Triton-X100. For H&E histology, the lungs were filled with 4% PFA via the trachea, dissected from the thorax, and placed into 4% paraformaldehyde (PFA). For the qPCR experiments, 6 mice . Blocking was performed using 1% BSA in PBS. The serum samples and conjugates were also added in 1% BSA in PBS. Following the incubation of the serum samples, to detect specific IgGa, IgG2a, or IgA production, the plates were further processed with anti-mouse IgG1 (clone RMG1-1), anti-mouse IgG2a (clone RMG2a-62), or anti-mouse IgA (clone RMA-1) labeled with biotin, respectively, at a 1:1000 dilution  for 2 h at room temperature. After 2 h of incubation at room temperature with primary antibodies, incubation with streptavidin–HRP (BioLegend) at a 1:7000 dilution was performed for 1 h.For the total IgE measurement, plates were coated overnight with unlabeled anti-mouse IgE  at a 1 µg/mL concentration. Blocking with 1% BSA in PBS was performed. After that, the plates were incubated overnight with serum samples at different dilutions and processed with biotin-labeled anti-mouse IgEa . Incubation with a biotin-labeled conjugate was performed at room temperature for 3 h. In the final stage, incubation with streptavidin–HRP (BioLegend) at a 1:7000 dilution was performed for 1 h.The CXCL13 (BCA1) concentration in the lung homogenates was measured using a mouse BCA1 matched antibody pair kit  according to the manufacturer’s instructions.5 cells in 20 µL of PBS were added to 1 mL of ExtractRNA. The samples were incubated for 30 min at room temperature and then kept at −20 °C prior to RNA purification. 0.25 mL of chloroform was added to 1 mL of the sample during the RNA extraction. The samples were vortexed for 3 min at room temperature, after which they were centrifuged at 15,000× g for 15 min at 4 °C. The upper aqueous phase was transferred to a new 1.5 mL tube, and then, 600 µL of isopropanol was added to the sample. The samples were incubated for 30 min at −20 °C for precipitation and then centrifuged for 10 min at 12,000× g. After decanting the supernatant, 1 mL of 75% ethanol was carefully added to each sample, after which all the samples were centrifuged again for 10 min at 12,000× g. The supernatant was discarded, and the pellets were washed with ethanol again. After that, the samples were heated at 50 °C in uncapped 1.5 mL tubes for 3–5 min to dry up the remaining ethanol.RNA was extracted using the standard phenol–chloroform method, followed by RNAse-free DNAse treatment (Thermo Fisher Scientific). Briefly, small patches of lung tissue (about 3 mm × 3 mm × 2 mm) from each mouse were homogenized in 1 mL of commercial ExtractRNA solution  containing phenol. For the lymph nodes, 3 × 10To remove genomic DNA from our samples, we used RNAse-free DNAse I  according to the manufacturer’s protocol. The samples were diluted in 8 µL of DEPC-treated water with the addition of 1 µL of 10× reaction buffer and 1 µL of DNAse I (1U) solution. Incubation at 37 °C for 30 min was performed. To activate DNAse I without RNA degradation occurring in the presence of divalent cations, we added 1 µL of 50 mM EDTA and incubated the samples at 65 °C for 10 min. The remaining template was diluted in a 300 µL volume with DEPC water and kept at −20 °C before usage.For the measurement of DNA excision circles corresponding to direct and sequential IgE switches, we did not perform DNA digestion; rather, cDNA was synthesized using a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific).GAPDH and HPRT, and calculated as 2−Δ(ΔCt) compared to the expression in the tissues of intact mice. The data are shown as the relative expression, determined as the ratio of the 2−Δ(ΔCt) for the experimental groups to that for the intact mice. The reaction was performed in a CFX Connect Amplificator  according to the following protocol: 95 °C initial denaturation for 3 min followed by 50 cycles of 5 s denaturation at 95 °C and 20 s annealing and elongation at 64 °C. The reaction was performed in 96-well plates  in a 20 µL volume. The forward and reverse primer concentrations were both 0.4 µM, and the probe concentration was 0.2 µM. 3 µL of diluted template was used in each reaction. The primers and probes were designed using NIH Primer BLAST and synthesized with Evrogen . The primer and probe sequences used in the study are shown in Quantitative PCR (qPCR) was performed as previously described . Kits frn = 5 from each group) were randomly selected. BAL fluid was collected as described in The manifestation of allergic airway inflammation was estimated by the presence of myeloid cells in the BAL. For this, mice . The samples were fixed with 4% PFA and kept in it before the preparation of the histological sections. The tissue samples were dehydrated in solutions with increasing ethanol concentrations  for 45 min and then kept in xylene for 1 h. The tissue samples were then embedded in paraffin. Histological sections 8 nm thick were made on a microtome (Thermo Fisher Scientific HM 355S). The histological sections were rehydrated by incubating them twice for 5 min in a xylene solution and then twice in 100% ethanol for 5 min, followed by two incubations each in 95% ethanol and 70% ethanol for 5 min and, finally, two incubations in water. After filling them with 4% PFA and dissecting them from the thorax, the lung samples were cut at −20 °C on 20 µm-thick sections and were stained using an H&E staining kit  according to the manufacturer’s instructions. Microscopic images with different magnifications were obtained. In order to estimate the impact of i.n.-administered OVA and DEPs on inducible bronchus-associated lymphoid tissue (iBALT) induction and growth, we quantified the relative area as a ratio: Total area of all histological features/Total area of iBALT structures occupied by iBALTs in these sections.Lung tissue samples were subjected to H&E histology. A separate experiment was carried out to obtain samples for H&E histology  were included in the final analysis from independent experiments .The group means and standard deviations were quantified in each case. ANOVA with correction for multiple comparisons was used to evaluate the significance of the differences between groups. The normality of the distribution of the data in each group was assessed using a Shapiro–Wilk test, and the probability of a normal distribution for each group parameter included in the final analysis was >0.95. The ELISA, qPCR, and BAL cell analysis experiments were performed independently three times. The H&E histological experiments were performed twice. Differences with To evaluate the effects of DEPs on pro-allergic antibody response, we first measured the production of allergen-specific antibodies in mice. Our results show that both DEPs induced IgE-antibody formation at comparable levels when administered at high but not at low doses, so the ability of DEPs to induce IgE responses was relatively independent of the co-administered OVA doses. High doses of DEPs in combination with low A,B or hiThe DEPs also induced specific IgG1 formation in mice when co-administered with a low (0.3 µg) OVA dose, and the titer of the specific IgG1 was higher upon co-administration with the high (30 µg) OVA dose . In the High but not low doses of both types of DEPs stimulated total IgE production in combination with low and high OVA doses. Although the induction of total IgE when DEPs were administered without OVA, compared with the level in saline-administered mice, was not very high, it was statistically significant . The oveWe next evaluated whether DEP-mediated OVA-specific humoral immune responses resulted in OVA-specific sensitization and subsequent asthma development at an OVA-challenging dose. We focused on the effects of a DEP dose of 150 µg/mouse, since only this particular amount stimulated the formation of pro-allergic antibodies. Despite the comparable levels of specific IgE induced by both high and low OVA doses co-administered together with DEPs, significantly higher titers of IgG1 and IgG2a that accompanied IgE production were observed at high OVA doses . In steaThe administration of a challenging OVA dose appears to promote a multicellular immune response  only but not mature IgG1 producing cells markers (postswitch γ1) in both the lungs and the regional lymph nodes (germlineε) when administered with low OVA doses  in lung tissue after the administration of low OVA doses  and IgE production (postswitch ε transcripts) induced in response to DEPs in the OVA-immunized animals were more independent on the administered OVA dose and type of DEP. In the DEP1-treated low-OVA dose group, both direct (µ–ε) and sequential (γ1–ε) mechanisms mediated the local IgE class switch  expression independently of the co-administered OVA dose. OVA alone did not induce IFNα1 expression, at either a low or high dose  contributes to iBALT formation . High OVse group B. It wasigh dose C.IL-4 expression in lung tissue independently of the OVA dose . DEP1 dEbi2, a marker of extrafollicular focus formation [Both types of particles triggered the expression of ormation , and thiIt is generally accepted that DEPs induce the formation of pro-allergic antibodies and the subsequent development of asthma in humans and in laboratory animal asthma models ,21,22,23Despite such differences, the particles stimulated humoral immune responses at comparable levels. Although only DEP2 stimulated IgA formation, the resulting IgA titers were not very high in comparison with the IgG titers, and this phenomenon may not have very marked effects in relation to allergic inflammation. In relation to IgE, the effects of these particles on IgG1 and even IgG2a production were similar. While, in some cases, IgG1 and IgG2a production were stimulated by relatively low DEP doses, IgE production was only stimulated by high DEP doses . Although such amounts of DEPs usually do not penetrate into the human body, even in heavily polluted industrial or urban areas, it should be noted that DEPs tend to accumulate in the body over a long time and, therefore, reach high doses . Our datHowever, in the latter study, the allergen extract contained additional stimuli as opposed to pure protein . The facBcl6 in mice immunized with low doses alone or with low OVA doses with DEP1. Therefore, DEPs stimulate an OVA-specific pro-allergic humoral response over a wide range of OVA doses, in contrast to the situation observed in the adjuvant-free model in our previous work [We previously showed that upon the administration of antigen in fat tissue with tertiary lymphoid structures, mainly low but not high antigen doses induced IgE production . Howeverous work .Generally, DEPs stimulate the accumulation of three major types of inflammatory cells in BAL fluid in our model: eosinophils, macrophages, and neutrophils, as well as OVA-specific IgE and type-2 cytokines. Therefore, our DEP-induced asthma model is appropriate for the evaluation of the effects of potential therapeutics on the key parameters of allergic asthma linked to the type-2 immune response. In our asthma model, DEPs potentiated eosinophils’ accumulation after high-dose-OVA challenge when combined with OVA during eight weeks of immunization. The number of eosinophils in BAL was slightly higher in the mice immunized with a low (0.3 µg) OVA dose in combination with DEP1 than in the mice immunized with a high (30 µg) OVA dose, but the difference was not significant. On the other hand, high but not low OVA doses synergized with DEPs in the potentiation of macrophages’ accumulation. Generally, the number of macrophages was significantly higher than the number of eosinophils and neutrophils. All the particles induced significant expression of type-2 cytokine genes in the lungs. Therefore, we conclude that in our case, DEPs induced T-helper-2-type-associated asthma involving eosinophils, IL-4, and IL-13. We used high DEP doses in this study, which, according to , are morpostswitch ε transcripts accumulate in both the lungs and lymph nodes. This may be due to the migration of antigen-experienced B cells between two sites, as was shown by another research team for lung iBALT [DEPs stimulate both local and systemic IgE class switching when administered with low OVA doses. However, in the case of SRM1650B, we observed only local IgE class switching after their co-administration with a high OVA dose, and in the case of SRM2786, only the accumulation of local IgE-producing cells was observed after their co-administration with a high OVA dose. Despite the fact that only local IgE class switching was induced by DEPs, independent of the DEP type and OVA dose in OVA-immunized animals, ng iBALT  and as wng iBALT . germline and postswitch γ1 transcripts in some cases may reflect the fact that in the case of IgG1, DEPs stimulate its production without increasing IgG1 class switching per se but rather by accelerating the differentiation of IgG1-expressing B cells to the final stages of IgG1-producing plasma cells. It is also possible that the effects of DEPs on IgG1 class switching and the accumulation of IgG1-producing cells are stronger and more significant in the early stages of the process.From our previous work, we concluded that in young allergic patients  and in lIt is tempting to speculate that the different abilities of the two types of particulate matter to induce iBALT formation and growth after accumulation in the lung tissue are due to their different tendencies to form relatively large (>10 µm) aggregates. This is despite the fact that prior to the administration to mice, when the DEPs were sonicated, lung microphotographs from H&E histology showed that large aggregates of particles were present in the lung tissue, especially in the case of DEP1. According to their characteristics as revealed by NIST, DEP2 particles, despite their larger size prior to aggregation, are less prone to forming large aggregates . Large agermline ε and germline γ1 transcripts’ expression and increased the amount of circular µ-ε, not only in the lung tissue but also in the regional lymph nodes in high-OVA-dose-immunized animals. DEP2 was more potent than DEP1 in the stimulation of Tnfa expression in the lungs in combination with high OVA doses. Therefore, if some Tregs’ responses were indeed induced by DEP2 in the high-dose-immunized mice, it may be due to the induction of a compensatory response at later stages that is dispensable and insignificant for the inhibition of general inflammation [DEP2 (SRM2786), in comparison with DEP1 (SRM1650b), was able to stimulate IgA production in mice immunized with high OVA doses. Though IgA class switching is usually associated with Tregs , DEP2, iammation . This sepostswitch ε and postswitch γ1 transcripts in both the lungs and lymph nodes in combination with low OVA doses, but DEP2 did not induce the accumulation of the former in the lymph nodes and the latter in lungs. This may indicate that in the case of DEP2, the migration of mature Ig-producing B cells between two sites is more restricted. Both types of particles in combination with low OVA doses induce proinflammatory cytokine expression in lungs at comparable levels, as well as the B-cell activation marker Ebi2 associated with the extrafollicular B-cell response [Ebi2 expression, which is an indicator of extrafollicular focus formation [TNFα and IFNα1 expression, as well as IL-4 and IL-13, but the expression of IFNγ was not induced. From these data, it becomes clear that DEPs induce local IgE production by the specific stimulation of type-2 immune responses; this is in agreement with most previous studies [IFNγ expression in the mouse lungs in our work. As mentioned above, the authors of [A more important observation is that in combination with low OVA doses, DEP2 but not DEP1 induced de novo iBALT formation and growth, despite their similar ability to induce IgE production. This difference is linked to their different abilities to stimulate initial CXCL13 production and germinal center responses when the particles are co-administered with low OVA doses. It is likely that particles from the fine type of particulate matter DEP2 adsorb the co-administered antigen on their surface and then penetrate the epithelial barrier with it. When these particles accumulate in tissue, the adsorbed antigen serves as a stronger signal for B and T cells than soluble antigen . Certainresponse . Howeverresponse . Howeverormation . Both ty studies ,20,21,23 studies  or their studies  are alsothors of  used a sTNFα and IFNα1 expression, when these particles were co-administered with low OVA doses, it did not lead to iBALT induction and growth, notwithstanding the fact that these cytokines have iBALT-promoting activity [The main conclusion we can draw from this work is that local IgE production in response to particulate matter is independent of the ability of the particulate matter to induce iBALT formation and growth. According to qPCR data, this local IgE response was also independent of germinal centers; this is in agreement with recent results obtained by other researchers that the local IgE class was mostly associated with the extrafollicular response but not with germinal centers ,58 and bactivity ,43,45; iactivity ,45. Our activity , who fouactivity . In our activity  is closeBcl6 inhibitors), and must be sensitive to specific inhibitors of pathways responsible for the formation of B-cell aggregates. Further work is required to clarify this matter.However, from our work, it becomes clear that local IgE production, surprisingly, does not always require de novo iBALT formation and growth, which does not mean that iBALT per se was not required for IgE B-cell class switching. We also cannot exclude the possibility that in the case of more frequent administration of DEPs (5 times a week instead of 2–3 times as in ), the reOverall, DEPs can induce local as well as systemic B-cell IgE class switching, which leads to allergic immune responses. However, local but not systemic IgE class switching in response to DEPs could be observed independent of the OVA dose and type of particle. This local IgE class switch occurs mainly through direct mechanisms, but low OVA doses can also stimulate a sequential switch. Only high DEP doses induce IgE production, so DEPs may be responsible for atopic disease development only when present constantly and in very high concentrations in the air, e.g., if someone lives in a region with a high road traffic burden, or in allergy-prone individuals. Local DEPs induced B-cell IgE class switching independent of their ability to induce the expression of CXCL13, germinal center development, and de novo iBALT formation and growth. Small iBALTs initially present in the lungs or other structures may be responsible for IgE production in this case. DEP-induced local B-cell IgE class switching was accompanied by extrafollicular B-cell activation and the induction of the expression of cytokines associated with type-2 immune responses in the absence of type-1 immune response activation.


Macaca mulatta), 23 common long-tailed (M. fascicularis fascicularis), and 6 Burmese long-tailed (M. fascicularis aurea) macaque populations in Thailand. We captured, anesthetized, and collected throat, buccal, and rectal swab specimens from the macaques. We screened swabs for Mycobacterium tuberculosis complex (MTBC) using insertion sequence 6110–specific nested PCR. We found higher MTBC prevalence at both population and individual levels among M. mulatta than M. fascicularis fascicularis macaques; all 3 M. fascicularis aurea macaque populations were positive for tuberculosis. We found that throat swab specimens provided the best sample medium for detecting MTBC. Our results showed no difference in MTBC prevalence between male and female animals, but a higher percentage of adults were infected than subadults and juveniles. Although we detected no association between frequency of human–macaque interaction and MTBC prevalence, bidirectional zoonotic transmission should be considered a possible public health concern.We identified tuberculosis in 1,836 macaques from 6 wild rhesus ( Mycobacterium tuberculosis bacteria, is an airborne chronic infectious disease with zoonotic potential found among both humans and wildlife  and long-tailed (M. fascicularis) macaques have commonly been used as animal models for TB drug and vaccine research because clinical signs and immune responses after MTB infection are similar to those in humans  and Burmese long-tailed (M. fascicularis aurea) macaques at 8°10′–12°24′N latitude within Thailand  of long-tailed macaques whose habitats now overlap with human habitats in Thailand. Although both rhesus and long-tailed macaques are widely used for TB research, they differ in their TB pathogenesis, progression, and bacterial burden  from 32 location-defined populations of 2 species of macaques (rhesus and long-tailed), including 2 subspecies of long-tailed macaques (common and Burmese), with distribution ranges in Thailand . https://us.virbac.com) mixed with 20–50 μg/kg body weight of (dex)medetomidine hydrochloride   . After wThe National Primate Research Center of Thailand-Chulalongkorn University (NPRCT-CU) Animal Care and Use Committees approved all animal procedures (protocol review no. 2075007). The Thailand Department of the National Parks, Wildlife and Plant Conservation approved the protocols for capturing and collecting specimens from macaques. https://www.serva.de) and extracted genomic DNA using an automated QIAsymphony Virus/Pathogen Mini Kit . We measured the concentration of extracted DNA using QIAGEN QIAxpert. We amplified extracted DNA using nested PCR with MTBC insertion sequence (IS) 6110–specific primers, according to a protocol developed for specimens from humans (2+ plus), and 2.0 μL each of 2.5 mmol deoxynucleoside triphosphate and 0.125 μL TaKaRa Ex Taq Hot .We incubated swab samples at 70°C for 1 h with 50 μL of 30 mg/mL lysozyme solution . For the first round of PCR, we set thermal cycling at 98°C for 1 min, followed by 30 cycles at 93°C for 20 s, 65°C for 30 s, 72°C for 1 min, and 72°C for 10 min. We amplified 1 μL of the first-round PCR product in the second round at 98°C for 1 min, followed by 30 cycles at 93°C for 20 s, 48°C for 30 s, 72°C for 30 s, and 72°C for 10 min. We visualized the 181-bp PCR products using 2% wt/vol agarose gel electrophoresis and stained them with Invitrogen SYBR Safe DNA gel stain (Thermo Fisher). We ran the PCR using Applied Biosystems Verti 96-Well Thermal Cyclers  and submitted the samples to Macrogen (https://www.macrogen.com) for DNA sequencing. We aligned nucleotide sequences with published M. tuberculosis sequences accessed from GenBank using MEGA X software  strain, serially diluted (1:10) from 1 ng to 1 fg (M. tuberculosis H37Rv that we could detect by nested PCR as the limit of detection (LOD) for this technique. We validated specificity of the MTBC nested PCR with IS6110-specific primers using granuloma lung tissues from a naturally  macaque . We conf2 tests, we analyzed associations between MTBC prevalence and macaque species and subspecies, sex and age, and between MTBC prevalence and frequency of human–macaque interaction. Using Pearson correlation analysis, we analyzed correlations between MTBC prevalence and bodily site  of specimen collection and used SPSS Statistics 28 for Mac  to analyze data. We used p≤0.05 to indicate statistical significance. We calculated prevalence rate and 95% CIs for differences in MTBC shedding stratified by species and subspecies, sex, and age of macaques. Using Pearson χM. tuberculosis–infected macaque all showed 100% similarity with the MTB H37Rv complete genome (GenBank accession no. NC_000962.3) (data not shown). A BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi) search using the NCBI-registered complete genomes indicated that the specific 181-bp nucleotide sequence obtained from the IS6110-specific nested PCR  could be detected in the MTBC. Thus, we interpreted the results from the IS6110-specific nested PCR in this study as positive for MTBC. The 181-bp nucleotide sequences from granuloma lung tissues and 19 nested PCR–positive samples from a naturally The optimized MTBC nested PCR conditions of the H37Rv DNA (ATCC27294) strain showed LOD values of 100 fg/μL in the first round (580-bp product) and 10 fg/μL in the second round (181-bp product) . Because2= 0.04; p = 0.01), 1,681 throat and rectal , and 1,681 buccal and rectal swab samples . However, when we analyzed only MTBC-positive results, we found significant correlations only between 152 throat and buccal , and 99 throat and rectal swab samples , but not between 45 buccal and rectal swab samples .We detected MTBC IS6110 DNA among all 3 (100%) Burmese long-tailed macaque populations, higher than for either rhesus  or common long-tailed  macaque populations . We foun2 = 253.95; p = 0.01) among rhesus  than Burmese long-tailed  and common long-tailed  macaques. Most MTBC-positive common long-tailed macaques inhabited the central region of Thailand at 13°02′–15°36′N latitude . When we considered data on MTBC prevalence by sex of positive case-patients, the difference between males  and females  was not significant . We also found no significant difference by swab collection site between percentages of MTBC-positive macaques and frequency of human interaction . We futher analyzed the data for any correlation between MTBC prevalence and age and sex. We stratified data into 3 groups on the basis of age estimated from dental eruption at the time of capture; we classified both male and female macaques >6 years as adults, 3–6 years as subadults, and <3 years as juveniles , 4. MTBC-ranging macaques. Because of low MTBC concentration in the collected specimens, most 580-bp band PCR amplifications in the first round were undetected; however, we could clearly identify the 181-bp PCR amplicons in agarose gel in the second round. LOD for the second-round nested PCR, 10 fg/μL, was 10-fold lower than in the first round, 100 fg/μL, similar to findings reported elsewhere  is used globally to detect TB in macaques. Testers intradermally inject tuberculin antigens at the edge of the animal’s upper eyelid and look for an immune response (erythema or edema) at 24, 48, and 72 hours after injection (In a previous report, laboratory-tested rhesus macaques were more susceptible to MTBC infection than long-tailed macaques (In light of usual rates of MTBC infection among long-tailed macaques, it is notable that all 3 populations of Burmese long-tailed macaques were MTBC positive, in spite of less frequent contact with humans, rare or monthly, than for populations of common long-tailed macaques. In particular, the World War Museum and Mangrove Forest Research Center populations in Ranong Province in southern Thailand reside in mangrove forests and roam freely for invertebrate foods. Unique genetic characteristics among Burmese long-tailed macaques, identified through intensive genetic studies (Mycobacterium spp. were not detected in any of 649 free-living common long-tailed macaques from 26 locations (MTBC shedding in 10/23 common long-tailed macaques populations in our study differed from a report published elsewhere (Although nested PCR is highly sensitive and less time-consuming than other methods, it can detect TB only during the active shedding stage of infection (Because MTBC-infected wild macaques could be reservoirs of the pathogen and it might be transmitted back to humans, bidirectional zoonotic transmission should be considered, especially for populations in which the MTBC-infected macaques interact with humans on a daily basis. In a worst-case scenario, MTBC bacteria could mutate after infecting a macaque, then be transmitted back to humans; existing commercial antimycobacterial agents might be ineffective for treating the mutated pathogen (