LetB Structure Reveals a Tunnel for Lipid Transport across the Bacterial Envelope.

Gram-negative bacteria are surrounded by an outer membrane composed of phospholipids and lipopolysaccharide, which acts as a barrier and contributes to antibiotic resistance. The systems that mediate phospholipid trafficking across the periplasm, such as MCE (Mammalian Cell Entry) transporters, have not been well characterized. Our ~3.5 Å cryo-EM structure of the E. coli MCE protein LetB reveals an ~0.6 megadalton complex that consists of seven stacked rings, with a central hydrophobic tunnel sufficiently long to span the periplasm. Lipids bind inside the tunnel, suggesting that it functions as a pathway for lipid transport. Cryo-EM structures in the open and closed states reveal a dynamic tunnel lining, with implications for gating or substrate translocation. Our results support a model in which LetB establishes a physical link between the two membranes and creates a hydrophobic pathway for the translocation of lipids across the periplasm.

Architectures of Lipid Transport Systems for the Bacterial Outer Membrane.

How phospholipids are trafficked between the bacterial inner and outer membranes through the hydrophilic space of the periplasm is not known. We report that members of the mammalian cell entry (MCE) protein family form hexameric assemblies with a central channel capable of mediating lipid transport. The E. coli MCE protein, MlaD, forms a ring associated with an ABC transporter complex in the inner membrane. A soluble lipid-binding protein, MlaC, ferries lipids between MlaD and an outer membrane protein complex. In contrast, EM structures of two other E. coli MCE proteins show that YebT forms an elongated tube consisting of seven stacked MCE rings, and PqiB adopts a syringe-like architecture. Both YebT and PqiB create channels of sufficient length to span the periplasmic space. This work reveals diverse architectures of highly conserved protein-based channels implicated in the transport of lipids between the membranes of bacteria and some eukaryotic organelles.

MceG stabilizes the Mce1 and Mce4 transporters in Mycobacterium tuberculosis.

Lipids are important nutrients for Mycobacterium tuberculosis (Mtb) to support bacterial survival in mammalian tissues and host cells. Fatty acids and cholesterol are imported across the Mtb cell wall via the dedicated Mce1 and Mce4 transporters, respectively. It is thought that the Mce1 and Mce4 transporters are comprised of subunits that confer substrate specificity and proteins that couple lipid transport to ATP hydrolysis, similar to other bacterial ABC transporters. However, unlike canonical bacterial ABC transporters, Mce1 and Mce4 appear to share a single ATPase, MceG. Previously, it was established that Mce1 and Mce4 are destabilized when key transporter subunits are rendered nonfunctional; therefore, we investigated here the role of MceG in Mce1 and Mce4 protein stability. We determined that key residues in the Walker B domain of MceG are required for the Mce1- and Mce4-mediated transport of fatty acids and cholesterol. Previously, it has been established that Mce1 and Mce4 are destabilized and/or degraded when key transporter subunits are rendered nonfunctional, thus we investigated a role for MceG in stabilizing Mce1 and Mce4. Using an unbiased quantitative proteomic approach, we demonstrate that Mce1 and Mce4 proteins are specifically degraded in mutants lacking MceG. Furthermore, bacteria expressing Walker B mutant variants of MceG failed to stabilize Mce1 and Mce4, and we show that deleting MceG impacts the fitness of Mtb in the lungs of mice. Thus, we conclude that MceG represents an enzymatic weakness that can be potentially leveraged to disable and destabilize both the Mce1 and Mce4 transporters in Mtb.

ATP disrupts lipid-binding equilibrium to drive retrograde transport critical for bacterial outer membrane asymmetry.

The hallmark of the gram-negative bacterial envelope is the presence of the outer membrane (OM). The OM is asymmetric, comprising lipopolysaccharides (LPS) in the outer leaflet and phospholipids (PLs) in the inner leaflet; this critical feature confers permeability barrier function against external insults, including antibiotics. To maintain OM lipid asymmetry, the OmpC-Mla system is believed to remove aberrantly localized PLs from the OM and transport them to the inner membrane (IM). Key to the system in driving lipid trafficking is the MlaFEDB ATP-binding cassette transporter complex in the IM, but mechanistic details, including transport directionality, remain enigmatic. Here, we develop a sensitive point-to-point in vitro lipid transfer assay that allows direct tracking of [14C]-labeled PLs between the periplasmic chaperone MlaC and MlaFEDB reconstituted into nanodiscs. We reveal that MlaC spontaneously transfers PLs to the IM transporter in an MlaD-dependent manner that can be further enhanced by coupled ATP hydrolysis. In addition, we show that MlaD is important for modulating productive coupling between ATP hydrolysis and such retrograde PL transfer. We further demonstrate that spontaneous PL transfer also occurs from MlaFEDB to MlaC, but such anterograde movement is instead abolished by ATP hydrolysis. Our work uncovers a model where PLs reversibly partition between two lipid-binding sites in MlaC and MlaFEDB, and ATP binding and/or hydrolysis shift this equilibrium to ultimately drive retrograde PL transport by the OmpC-Mla system. These mechanistic insights will inform future efforts toward discovering new antibiotics against gram-negative pathogens.

Integrating meteorological and geospatial data for forest fire risk assessment.

Understanding the factors that cause fire is crucial for minimizing the fire risk. In this research, a comprehensive approach was adopted to recognize factors influencing forest fires. Golestan National Park (GNP) was considered as a representative area with a humid climate in this study. Initially, using the Multi-Criteria Evaluation Method, a fire risk map was created by analyzing natural and human factors, and vulnerable areas were identified. Then, the relationship between key elements such as meteorological conditions, Land Surface Temperature (LST), and precipitation, with the occurrence of fire in different years was investigated. CHIRPS and Landsat data were utilized to assess LST changes and precipitation. 23-year changes in fire occurrence areas in GNP were acquired using MODIS products. The results of the data analysis showed that the highest number of fires occurred in forest areas, and in the fire risk prediction map, the extremely high-risk class is completely consistent with the ground truth data. The assigned weights, derived from expert opinions, highlight the substantial significance of elevation, and distance from roads and settlements. Additionally, the effectiveness of the model in providing reliable forecasts for fire risks in GNP is highlighted by the ROC curve with an AUC value of 0.83. Forest fires within GNP exhibit a distinct seasonality, predominantly occurring from July to December. During the warmer months, by coinciding with summer excursions, human activities may contribute to the ignition of fires. In 2013 and 2014, rising fire incidents correlated with elevated temperatures, hinting at a potential connection. GNP fires showed an upward trend with higher monthly LST and a downward trend with increased annual precipitation. The results showed that there is a relationship between LST, precipitation, and the occurrence of fire in GNP. Approximately 176.15 ha of GNP's forest areas have been destroyed by fires over the last two decades. This research demonstrated that there is a dynamic interaction between environmental conditions and fire incidents. By considering these factors, managers and environmental planners can develop effective strategies for managing and preventing forest fire risks.

Mycobacterium tuberculosis Mce2D protein blocks M1 polarization in macrophages by inhibiting the ERK signaling pathway.

Macrophages play a pivotal role in controlling Mycobacterium infection, and the pathogen thrives in the event of immune evasion and immunosuppression of macrophages. Mammalian cell entry proteins (Mce) are required for Mycobacterium tuberculosis (M. tb) growth and the host cell's initial phagocytosis and cytokine response. Mce2D protein is one of a family of proteins that infect M. tb; however, the function and mechanism of action remain unclear. In this study, we constructed the Mce2D knockout strain using Mycobacterium smegmatis to study the function of Mce2D in the infection of macrophages. The results indicated that compared to the knockout strain, the release of proinflammatory cytokines (TNF-α and IL-1ß) reduced when WT strain infected the macrophages. Moreover, Mce2D boosted the metabolism of oxidized fatty acids, increased the energy supply of TCA, and lowered the glycolysis of glucose in macrophages after bacterial infection, all of which prevented the polarization of macrophages to M1, which was driven by the fact that Mce2D blocked ERK2 phosphorylation by interacting with ERK2 through its DEF motif. This, in turn, promoted nuclear translocation of HIF-1α, allowing signal accumulation, which increased the HIF-1α transcription levels. Finally, the mouse infection experiment showed that Mce2D caused blockage of M1 polarization of alveolar macrophages, resulting in reduced bactericidal activity and antigen presentation, weakening Th1 cell-mediated immune response and helping bacteria escape the immune system. Our results reveal that Mce2D causes immune escape by blocking M1 polarization in macrophages, providing potential targets for the rational design of therapies against M. tb infection.

Expression of mammalian cell entry genes in clinical isolates of M. tuberculosis and the cell entry potential and immunological reactivity of the Rv0590A protein.

Mammalian cell entry (mce) operons play a vital role in cell invasion and survival of M. tuberculosis. Of the mce genes, the function of Rv0590A is still unknown. The present study was performed to investigate the function and immunogenic properties of the protein Rv0590A. Human leukemia monocytic cell line (THP-1) derived macrophages were infected with M. tuberculosis H37Rv at 3, 6, and 24 h of infection. The maximum colony forming units (CFU) were observed at 6 h (p < 0.005), followed by 3 h after infection. M. tuberculosis H37Rv and clinical isolates representative of Delhi/CAS, EAI, Beijing, Haarlem and Euro-American-superlineage were included in the study for expression analysis of mce1A, mce2A, mce3A, mce4A, and Rv0590A genes. Maximum upregulation of all mce genes was observed at 3 h of infection. All the five clinical isolates and H37Rv upregulated Rv0590A at various time points. Macrophage infection with M. tuberculosis H37Rv-overexpressing Rv0590A gene showed higher intracellular CFU as compared to that of wild-type H37Rv. Further, purified Rv0590A protein stimulated the production of TNFα, IFNγ, and IL-10 in macrophages. Thus, Rv0590A was found to be involved in cell invasion and showed good immunological response.

Immuno-reactivity evaluation of Mce-truncated subunit candidate vaccine against Mycobacterium avium subspecies paratuberculosis challenge in the goat models.

BACKGROUND: Detection of an appropriate antigen with high immunogenicity can be a big step in the production of an effective vaccine for control of Johne's disease (JD). The aim of this study was to evaluate the efficacy of Mce-truncated protein as a subunit vaccine candidate for the control of JD in experimentally challenged goats. MATERIALS AND METHODS: Six healthy goat kids were immunized with Mce-truncated protein, and two goats were kept as controls. All kids were twice challenged orally with live Mycobacterium avium subspecies paratuberculosis(MAP) strain and half the goats from both the categories were sacrificed at 7 and 10 months after start of challenge study. Culture of MAP was performed from all the necropsied tissues to determine the true JD infection status. RESULTS: Mce-truncated protein only reacted with pooled vaccinated goat sera in western-blot. A significant increase in humoral immune response against Mce protein was also observed in vaccinated goats. Compared to the control group, vaccinated goats gained higher body weights and none of them shed MAP or showed histopatological lesions or colonization of MAP in their necropsy tissues. CONCLUSIONS: The new Mce protein based vaccine provided significant immunity in goats as they could meet the challenge with live MAP bacilli. Although the vaccine used in this study showed the high potential as a new effective vaccine for the control of JD, further validation study is still required to successfully implement the vaccine for JD control program.

Mce1R of Mycobacterium tuberculosis prefers long-chain fatty acids as specific ligands: a computational study.

The mce1 operon of Mycobacterium tuberculosis, which codes the Mce1 transporter, facilitates the transport of fatty acids. Fatty acids are one of the major sources for carbon and energy for the pathogen during its intracellular survival and pathogenicity. The mce1 operon is transcriptionally regulated by Mce1R, a VanR-type regulator, which could bind specific ligands and control the expression of the mce1 operon accordingly. This work reports computational identification of Mce1R-specific ligands. Initially by employing cavity similarity search algorithm by the ProBis server, the cavities of the proteins similar to that of Mce1R and the bound ligands were identified from which fatty acids were selected as the potential ligands. From the earlier-generated monomeric structure, the dimeric structure of Mce1R was then modeled by the GalaxyHomomer server and validated computationally to use in molecular docking and molecular dynamics simulation analysis. The fatty acid ligands were found to dock within the cavity of Mce1R and the docked complexes were subjected to molecular dynamics simulation to explore their stabilities and other dynamic properties. The data suggest that Mce1R preferably binds to long-chain fatty acids and undergoes distinct structural changes upon binding.

Mapping flood vulnerability using an analytical hierarchy process (AHP) in the Metropolis of Mumbai.

The burgeoning significance of urban floods in the context of evolving climate dynamics and shifting rainfall patterns underscores the exigency for comprehensive investigation and mitigation strategies. The study employs a multi-criteria assessment (MCE) approach and the analytical hierarchy process (AHP) to evaluate flood-vulnerable zones, wards, and sub-category-wise flood locations in Greater Mumbai. The AHP technique is used to evaluate flood-vulnerable impacting parameters such as rainfall (29.42%), slope (20.96%), land use/land cover (17.52%), vicinity to sewers and storm-water drainage (13.99%), vicinity to natural drainage (8.97%), vegetation (5.58%), and soil (3.56%). The study area is classified under different vulnerable categories as severe vulnerable (46.72%), high to very high (18.74%), and slight to moderate (34.54%). Researchers analysed 234 waterlogged locations, revealing that 85.46% (200 locations) were in the severe to very high vulnerability category, and only 14.52% (34 locations) were in the other three categories. Flood locations are more affected by slope (under the categories of < 5 m and 5.01-10 m), built-up land, sewers and storm water drainage (< 125 m), natural drainage (< 250 m), rainfall (< 2000 to 2200 mm), lowest dense vegetation, and coastal alluvium in soils. These model-based flood vulnerability maps are crucial for planning flood conservation and mitigation measures.

Ordinal Conditional Entropy Displays Reveal Intrinsic Characteristics of the Rosenberg Self-Esteem Scale.

Individual subjects' ratings neither are metric nor have homogeneous meanings, consequently digital- labeled collections of subjects' ratings are intrinsically ordinal and categorical. However, in these situations, the literature privileges the use of measures conceived for numerical data. In this paper, we discuss the exploratory theme of employing conditional entropy to measure degrees of uncertainty in responding to self-rating questions and that of displaying the computed entropies along the ordinal axis for visible pattern recognition. We apply this theme to the study of an online dataset, which contains responses to the Rosenberg Self-Esteem Scale. We report three major findings. First, at the fine scale level, the resultant multiple ordinal-display of response-vs-covariate entropy measures reveals that the subjects on both extreme labels (high self-esteem and low self-esteem) show distinct degrees of uncertainty. Secondly, at the global scale level, in responding to positively posed questions, the degree of uncertainty decreases for increasing levels of self-esteem, while, in responding to negative questions, the degree of uncertainty increases. Thirdly, such entropy-based computed patterns are preserved across age groups. We provide a set of tools developed in R that are ready to implement for the analysis of rating data and for exploring pattern-based knowledge in related research.

Drug Degradation Caused by mce3R Mutations Confers Contezolid (MRX-I) Resistance in Mycobacterium tuberculosis.

Contezolid (MRX-I), a safer antibiotic of the oxazolidinone class, is a promising new antibiotic with potent activity against Mycobacterium tuberculosis (MTB) both in vitro and in vivo. To identify resistance mechanisms of contezolid in MTB, we isolated several in vitro spontaneous contezolid-resistant MTB mutants, which exhibited 16-fold increases in the MIC of contezolid compared with the parent strain but were still unexpectedly susceptible to linezolid. Whole-genome sequencing revealed that most of the contezolid-resistant mutants bore mutations in the mce3R gene, which encodes a transcriptional repressor. The mutations in mce3R led to markedly increased expression of a monooxygenase encoding gene Rv1936. We then characterized Rv1936 as a putative flavin-dependent monooxygenase that catalyzes the degradation of contezolid into its inactive 2,3-dihydropyridin-4-one (DHPO) ring-opened metabolites, thereby conferring drug resistance. While contezolid is an attractive drug candidate with potent antimycobacterial activity and low toxicity, the occurrence of mutations in Mce3R should be considered when designing combination therapy using contezolid for treating tuberculosis.

Microchip electrophoresis separation coupled to mass spectrometry (MCE-MS) for the rapid monitoring of multiple quality attributes of monoclonal antibodies.

Therapeutic monoclonal antibodies (mAbs) are highly heterogeneous as a result of posttranslational modifications (PTMs) during bioprocessing and storage. The modifications that impact mAb product quality are regarded as critical quality attributes and require monitoring. The conventional LC-mass spectrometer (MS) method used for product quality monitoring may require protein A purification prior to analysis. In this paper, we present a high-throughput microchip electrophoresis (<4 min) in-line with MS (MCE-MS) that enables baseline separation and characterization of Fc, Fd', and light chain (LC) domains of IdeS-treated mAb sample directly from bioreactor. The NISTmAb was used to optimize the MCE separation and to assess its capability of multiple attribute monitoring. The MCE-MS can uniquely separate and characterize deamidated species at domain level compared to LC-MS method. Two case studies were followed to demonstrate the method capability of monitoring product quality of mAb samples from stability studies or directly from bioreactors.

The Immediate and Sustained Effects of Exercise-Induced Hemodynamic Response on Executive Function During Fine Motor-Cognitive Tasks Using Functional Near-Infrared Spectroscopy.

BACKGROUND: Several studies have shown that acute exercise has a small positive effect on cognitive performance. However, it is still unclear what type of exercise has a sustained impact on cognitive performance during post-exercise recovery. Therefore, the purpose of our study was to investigate cognitive performance at the behavioral level, and their neural correlates after a 10-minute post-exercise recovery period with two different types of exercise intervention (high-intensity interval exercise (HIIE) vs. Moderate-intensity continuous exercise (MCE)). METHODS: A total of 29 healthy young adults (7 women) between the ages of 19 and 33 with fair to good cardiovascular fitness were submitted to two different exercise protocols and a recovery session. Cognitive function was assessed using a digital Trail-Making-Test (dTMT). Cortical activity in the prefrontal and the motor cortex using functional near-infrared spectroscopy (fNIRS) was measured before, after acute exercise, and during recovery. The statistical analysis of fNIRS data was performed by comparing the slope and mean of the hemodynamic response. RESULTS: High levels of hemodynamic responses were observed in the prefrontal and motor cortex on the brain during performing the dTMT while walking from pre- to post-exercise and decreased again in post-recovery, accompanied by improvement and maintenance of cognitive performance. Notably, a high hemodynamic response in the left motor area of the brain was maintained by HIIE in post-recovery compared with MCE. CONCLUSIONS: The high cortical activation in the left motor area from post-exercise to recovery for the HIIE group may be due to the additional availability of neural resources for fine motor and postural control by high-intensity exercise-induced fatigue. Additionally, the improved cognitive performance may have effectively utilized the available neural resources in the frontal lobe, depending on the condition (sitting and walking) and the two types of exercise protocol (HIIE and MCE).

Evidence for the Mycobacterial Mce4 Transporter Being a Multiprotein Complex.

Mycobacteria possess Mce transporters that import lipids and are thought to function analogously to ATP-binding cassette (ABC) transporters. However, whereas ABC transporters import substrates using a single solute-binding protein (SBP) to deliver a substrate to permease proteins in the membrane, mycobacterial Mce transporters have a potential for six SBPs (MceA to MceF) working with a pair of permeases (YrbEA and YrbEB), a cytoplasmic ATPase (MceG), and multiple Mce-associated membrane (Mam) and orphaned Mam (Omam) proteins to transport lipids. In this study, we used the model mycobacterium Mycobacterium smegmatis to study the requirement for individual Mce, Mam, and Omam proteins in Mce4 transport of cholesterol. All of the Mce4 and Mam4 proteins we investigated were required for cholesterol uptake. However, not all Omam proteins, which are encoded by genes outside mce loci, proved to contribute to cholesterol import. OmamA and OmamB were required for cholesterol import, while OmamC, OmamD, OmamE, and OmamF were not. In the absence of any single Mce4, Mam4, or Omam protein that we tested, the abundance of Mce4A and Mce4E declined. This relationship between the levels of Mce4A and Mce4E and these additional proteins suggests a network of interactions that assemble and/or stabilize a multiprotein Mce4 transporter complex. Further support for Mce transporters being multiprotein complexes was obtained by immunoprecipitation-mass spectrometry, in which we identified every single Mce, YrbE, MceG, Mam, and Omam protein with a role in cholesterol transport as associating with Mce4A. This study represents the first time any of these Mce4 transporter proteins has been shown to associate.IMPORTANCE How lipids travel between membranes of diderm bacteria is a challenging mechanistic question because lipids, which are hydrophobic molecules, must traverse a hydrophilic periplasm. This question is even more complex for mycobacteria, which have a unique cell envelope that is highly impermeable to molecules. A growing body of knowledge identifies Mce transporters as lipid importers for mycobacteria. Here, using protein stability experiments and immunoprecipitation-mass spectrometry, we provide evidence for mycobacterial Mce transporters existing as multiprotein complexes.

Recent advances in nanomaterial-assisted detection coupled with capillary and microchip electrophoresis.

Nanomaterials have drawn much attention because of their unique properties enabling them to play important roles in various applications in different areas. This review covers literature data in the Web of Science from January 2017 to August 2020, focusing on the applications of nanomaterials (nanoparticles, quantum dots, nanotubes, and graphene) in CE and MCE to achieve enhanced sensitivity of several detection techniques: fluorescence, colorimetry, amperometry, and chemiluminescence /electrochemiluminescence. For the articles surveyed, the types of nanomaterials used, detection mechanisms, analytical performance, and applications are presented and discussed.

Suitability Analysis of Acoustic Refugia for Endangered Killer Whales (Orcinus orca) Using the GIS-based Logic Scoring of Preference Method.

An emerging priority in marine noise pollution research is identifying marine "acoustic refugia" where noise levels are relatively low and good-quality habitat is available to acoustically sensitive species. The endangered Southern Resident population of killer whales (Orcinus orca) that inhabits the transboundary Salish Sea in Canada and the USA are affected by noise pollution. Geographic Information Systems (GIS) and spatial multicriteria evaluation (MCE) methods have been used to operationalize suitability analysis in ecology and conservation for site selection problems. However, commonly used methods lack the ability to represent complex logical relationships between input criteria. Therefore, the objective of this study is to apply a more advanced MCE method, known as Logic Scoring of Preference (LSP), to identify acoustic refugia for killer whales in the Salish Sea. This GIS-based LSP-MCE approach considers multiple input criteria by combining input data representing killer whale habitat requirements with noise pollution and other factors to identify suitable acoustic refugia. The results indicate the locations of suitable acoustic refugia and how they are affected by noise pollution from marine vessels in three scenarios developed to represent different levels of vessel traffic. Identifying acoustic refugia can contribute to efforts to reduce the effect of marine noise pollution on killer whale populations by highlighting high-priority areas in which to implement policies such as traffic-limiting measures or marine protected areas. Moreover, the proposed LSP-MCE procedure combines criteria in a stepwise manner that can support environmental management decision-making processes and can be applied to other marine suitability analysis contexts.

Presentation of landscape-fuzzy approach of forest capability evaluation (LFAFCE) for degraded sites.

Evaluation of forest sites capability (EFSC) is important for the restoration of degraded areas. The current costly EFSC approaches developed based on forest stand structure criteria is too costly for less developed countries (LDC) and not suitable for severely degraded lands. This paper describes an inexpensive Landscape-fuzzy approach for forest capability evaluation (LFAFCE) that can be used to restore degraded forest areas especially in LDC. Five physical criteria of slope, hillshade, altitude, precipitation, and geo formation were evaluated in the Zagros region of western Iran using the fuzzy membership functions, prioritized by analytic network process (ANP), and combined with GIS-based weighted linear combination. We then performed multi-criteria evaluation integrated by GIS. Given the positive correlation between the independent variable of EFSC and the dependent variable of the dominant tree height, the model results were validated based on the linear regression of the relationship between the two variables. The results of the validation showed that the linear regression model with appropriate coefficient of determination was significant. The results of EFSC by LFAFCE showed that most of the forest area was allocated to two classes: well (75%) and moderate (21.8%). In total, only 3.2% of the area belonged to the marginal (0.4%), high (0.1%), and unsuitable regions (2.7%) classes. Our results demonstrate that LFAFCE is valid for low-cost evaluation of degraded area in Zagros and for other similar areas, if calibrated, where normal forest mass parameters are not available.

Recent advances in microchip enantioseparation and analysis.

This review summarizes recent developments (over the past decade) in the field of microfluidics-based solutions for enantiomeric separation and detection. The progress in various formats of microchip electrodriven separations, such as MCE, microchip electrochromatography, and multidimensional separation techniques, is discussed. Innovations covering chiral stationary phases, surface coatings, and modification strategies to improve resolution, as well as integration with detection systems, are reported. Finally, combinations with other microfluidic functional units are also presented and highlighted.

An examination of MCE filter morphology and implications on preparation and analysis of air samples for asbestos.

Morphological imperfections of mixed-cellulose ester filters and their possible influence on sample preparation and analysis of asbestos were examined. Filters were identified with large regions of non-porous "dead zones" which could negatively affect fiber deposition and, therefore, fiber recovery and analysis by Transmission Electron Microscopy (TEM). As these imperfections are effectively erased during the preparation of the sample, they may not be readily observed by TEM. Un-collapsed filters as well as those collapsed using dimethylformamide (DMF) and two acetone techniques were examined. In order to minimize negative sampling and analytical bias, it is suggested that MCE samples be collapsed utilizing a 35% DMF solution, etched with a correctly calibrated low-temperature etcher, carbon coated using a rotating and tilted stage, and analyzed with a strong analyst-independent grid square opening randomization scheme.