BACKGROUND: Immune cells that infiltrate lesions are important for atherosclerosis progression and immunotherapies. This study was aimed at gaining important new insights into the heterogeneity of these cells by integrating the sequencing results of multiple samples and using an enhanced single-cell sequencing workflow to overcome the limitations of a single study. RESULTS: Integrative analyses identified 28 distinct subpopulations based on gene expression profiles. Further analysis demonstrated that these cells manifested high heterogeneity at the levels of tissue preferences, genetic perturbations, functional variations, immune dynamics, transcriptional regulators, metabolic changes, and communication patterns. Of the T cells, interferon-induced CD8+ T cells were involved in the progression of atherosclerosis. In contrast, proinflammatory CD4+ CD28null T cells predicted a poor outcome in atherosclerosis. Notably, we identified two subpopulations of foamy macrophages that exhibit contrasting phenotypes. Among them, TREM2- SPP1+ foamy macrophages were preferentially distributed in the hypoxic core of plaques. These glycolytic metabolism-enriched cells, with impaired cholesterol metabolism and robust pro-angiogenic capacity, were phenotypically regulated by CSF1 secreted by co-localised mast cells. Moreover, combined with deconvolution of the bulk datasets, we revealed that these dysfunctional cells had a higher proportion of ruptured and haemorrhagic lesions and were significantly associated with poor atherosclerosis prognoses. CONCLUSIONS: We systematically explored atherosclerotic immune heterogeneity and identified cell populations underlying atherosclerosis progression and poor prognosis, which may be valuable for developing new and precise immunotherapies.

BACKGROUND: NorQ, a member of the MoxR-class of AAA+ ATPases, and NorD, a protein containing a Von Willebrand Factor Type A (VWA) domain, are essential for non-heme iron (FeB) cofactor insertion into cytochrome c-dependent nitric oxide reductase (cNOR). cNOR catalyzes NO reduction, a key step of bacterial denitrification. This work aimed at elucidating the specific mechanism of NorQD-catalyzed FeB insertion, and the general mechanism of the MoxR/VWA interacting protein families. RESULTS: We show that NorQ-catalyzed ATP hydrolysis, an intact VWA domain in NorD, and specific surface carboxylates on cNOR are all features required for cNOR activation. Supported by BN-PAGE, low-resolution cryo-EM structures of NorQ and the NorQD complex show that NorQ forms a circular hexamer with a monomer of NorD binding both to the side and to the central pore of the NorQ ring. Guided by AlphaFold predictions, we assign the density that "plugs" the NorQ ring pore to the VWA domain of NorD with a protruding "finger" inserting through the pore and suggest this binding mode to be general for MoxR/VWA couples. CONCLUSIONS: Based on our results, we present a tentative model for the mechanism of NorQD-catalyzed cNOR remodeling and suggest many of its features to be applicable to the whole MoxR/VWA family.

BACKGROUND: Undernourishment in utero has deleterious effects on the metabolism of offspring, but the mechanism of the transgenerational transmission of metabolic disorders is not well known. In the present study, we found that undernourishment in utero resulted in metabolic disorders of female F1 and F2 in mouse model. RESULTS: Undernutrition in utero induced metabolic disorders of F1 females, which was transmitted to F2 females. The global methylation in oocytes of F1 exposed to undernutrition in utero was decreased compared with the control. KEGG analysis showed that genes with differential methylation regions (DMRs) in promoters were significantly enriched in metabolic pathways. The altered methylation of some DMRs in F1 oocytes located at the promoters of metabolic-related genes were partially observed in F2 tissues, and the expressions of these genes were also changed. Meanwhile, the abnormal DNA methylation of the validated DMRs in F1 oocytes was also observed in F2 oocytes. CONCLUSIONS: These results indicate that DNA methylation may mediate the transgenerational inheritance of metabolic disorders induced by undernourishment in utero via female germline.

BACKGROUND: The nuclease NurA and the ATPase/translocase HerA play a vital role in repair of double-strand breaks (DSB) during the homologous recombination in archaea. A NurA-HerA complex is known to mediate DSB DNA end resection, leading to formation of a free 3' end used to search for the homologous sequence. Despite the structures of individual archaeal types of NurA and HerA having been reported, there is limited information regarding the molecular mechanisms underlying this process. Some bacteria also possess homologs of NurA and HerA; however, the bacterial type of this complex, as well as the detailed mechanisms underlying the joining of NurA-HerA in DSB DNA end resection, remains unclear. RESULTS: We report for the first time the crystal structures of Deinococcus radiodurans HerA (drHerA) in the nucleotide-free and ADP-binding modes. A D. radiodurans NurA-HerA complex structure was constructed according to a low-resolution cryo-electron microscopy map. We performed site-directed mutagenesis to map the drNurA-HerA interaction sites, suggesting that their interaction is mainly mediated by ionic links, in contrast to previously characterized archaeal NurA-HerA interactions. The key residues responsible for the DNA translocation activity, DNA unwinding activity, and catalytic activities of the drNurA-HerA complex were identified. A HerA/FtsK-specific translocation-related motif (TR motif) that guarantees the processivity of double-stranded DNA (dsDNA) translocation was identified. Moreover, a mechanism for the translocation-regulated resection of the 5' tail of broken dsDNA and the corresponding generation of a recombinogenic 3' single-stranded DNA tail by the drNurA-HerA complex was elucidated. CONCLUSIONS: Our work provides new insights into the mechanism underlying bacterial NurA-HerA-mediated DSB DNA end resection, and the way this complex digests the 5' tail of a DNA duplex and provides long 3' free end for strand invasion in the bacterial homologous recombination process.

BACKGROUND: Despite the process of DNA replication being mechanistically highly conserved, the location of origins of replication (ORI) may vary from one tissue to the next, or between rounds of replication in eukaryotes, suggesting flexibility in the choice of locations to initiate replication. Lists of human ORI therefore vary widely in number and location, and there are currently no methods available to compare them. Here, we propose a method of detection of ORI based on somatic mutation patterns generated by the mutator phenotype of damaged DNA polymerase epsilon (POLE). RESULTS: We report the genome-wide localization of constitutive ORI in POLE-mutated human tumors using whole genome sequencing data. Mutations accumulated after many rounds of replication of unsynchronized dividing cell populations in tumors allow to identify constitutive origins, which we show are shared with high fidelity between individuals and tumor types. Using a Smith-Waterman-like dynamic programming approach, we compared replication origin positions obtained from multiple different methods. The comparison allowed us to define a consensus set of replication origins, identified consistently by multiple ORI detection methods. Many DNA features co-localized with the consensus set of ORI, including chromatin loop anchors, G-quadruplexes, S/MARs, and CpGs. Among all features, the H2A.Z histone exhibited the most significant association. CONCLUSIONS: Our results show that mutation-based detection of replication origins is a viable approach to determining their location and associated sequence features.

BACKGROUND: CRISPR-based screens are revolutionizing drug discovery as tools to identify genes whose ablation induces a phenotype of interest. For instance, CRISPR-Cas9 screening has been successfully used to identify novel therapeutic targets in cancer where disruption of genes leads to decreased viability of malignant cells. However, low-activity guide RNAs may give rise to variable changes in phenotype, preventing easy identification of hits and leading to false negative results. Therefore, correcting the effects of bias due to differences in guide RNA efficiency in CRISPR screening data can improve the efficiency of prioritizing hits for further validation. Here, we developed an approach to identify hits from negative CRISPR screens by correcting the fold changes (FC) in gRNA frequency by the actual, observed frequency of indel mutations generated by gRNA. RESULTS: Each gRNA was coupled with the "reporter sequence" that can be targeted by the same gRNA so that the frequency of mutations in the reporter sequence can be used as a proxy for the endogenous target gene. The measured gRNA activity was used to correct the FC. We identified indel generation efficiency as the dominant factor contributing significant bias to screening results, and our method significantly removed such bias and was better at identifying essential genes when compared to conventional fold change analysis. We successfully applied our gRNA activity data to previously published gRNA screening data, and identified novel genes whose ablation could synergize with vemurafenib in the A375 melanoma cell line. Our method identified nicotinamide N-methyltransferase, lactate dehydrogenase B, and polypyrimidine tract-binding protein 1 as synergistic targets whose ablation sensitized A375 cells to vemurafenib. CONCLUSIONS: We identified the variations in target cleavage efficiency, even in optimized sgRNA libraries, that pose a strong bias in phenotype and developed an analysis method that corrects phenotype score by the measured differences in the targeting efficiency among sgRNAs. Collectively, we expect that our new analysis method will more accurately identify genes that confer the phenotype of interest.

BACKGROUND: Perceptual learning refers to an augmentation of an organism's ability to respond to external stimuli, which has been described in most sensory modalities. Visual perceptual learning (VPL) is a manifestation of plasticity in visual information processing that occurs in the adult brain, and can be used to ameliorate the ability of patients with visual defects mainly based on an improvement of detection or discrimination of features in visual tasks. While some brain regions such as the primary visual cortex have been described to participate in VPL, the way more general high-level cognitive brain areas are involved in this process remains unclear. Here, we showed that the medial prefrontal cortex (mPFC) was essential for both the training and maintenance processes of VPL in mouse models. RESULTS: We built a new VPL model in a custom-designed training chamber to enable the utilization of miniScopes when mice freely executed the VPL task. We found that pyramidal neurons in the mPFC participate in both the training process and maintenance of VPL. By recording the calcium activity of mPFC pyramidal neurons while mice freely executed the task, distinct ON and OFF neural ensembles tuned to different behaviors were identified, which might encode different cognitive information. Decoding analysis showed that mouse behaviors could be well predicted using the activity of each ON ensemble. Furthermore, VPL recruited more reward-related components in the mPFC. CONCLUSION: We revealed the neural mechanism underlying vision improvement following VPL and identify distinct ON and OFF neural ensembles in the mPFC that tuned to different information during visual perceptual training. These results uncover an important role of the mPFC in VPL, with more reward-related components being also involved, and pave the way for future clarification of the reward signal coding rules in VPL.

BACKGROUND: Adaptation to high-altitude hypobaric hypoxia has been shown to require a set of physiological traits enabled by an associated set of genetic modifications, as well as transcriptome regulation. These lead to both lifetime adaptation of individuals to hypoxia at high altitudes and generational evolution of populations as seen for instance in those of Tibet. Additionally, RNA modifications, which are sensitive to environmental exposure, have been shown to play pivotal biological roles in maintaining the physiological functions of organs. However, the dynamic RNA modification landscape and related molecular mechanisms in mouse tissues under hypobaric hypoxia exposure remain to be fully understood. Here, we explore the tissue-specific distribution pattern of multiple RNA modifications across mouse tissues. RESULTS: By applying an LC-MS/MS-dependent RNA modification detection platform, we identified the distribution of multiple RNA modifications in total RNA, tRNA-enriched fragments, and 17-50-nt sncRNAs across mouse tissues; these patterns were associated with the expression levels of RNA modification modifiers in different tissues. Moreover, the tissue-specific abundance of RNA modifications was sensitively altered across different RNA groups in a simulated high-altitude (over 5500 m) hypobaric hypoxia mouse model with the activation of the hypoxia response in mouse peripheral blood and multiple tissues. RNase digestion experiments revealed that the alteration of RNA modification abundance under hypoxia exposure impacted the molecular stability of tissue total tRNA-enriched fragments and isolated individual tRNAs, such as tRNAAla, tRNAval, tRNAGlu, and tRNALeu. In vitro transfection experiments showed that the transfection of testis total tRNA-enriched fragments from the hypoxia group into GC-2spd cells attenuated the cell proliferation rate and led to a reduction in overall nascent protein synthesis in cells. CONCLUSIONS: Our results reveal that the abundance of RNA modifications for different classes of RNAs under physiological conditions is tissue-specific and responds to hypobaric hypoxia exposure in a tissue-specific manner. Mechanistically, the dysregulation of tRNA modifications under hypobaric hypoxia attenuated the cell proliferation rate, facilitated the sensitivity of tRNA to RNases, and led to a reduction in overall nascent protein synthesis, suggesting an active role of tRNA epitranscriptome alteration in the adaptive response to environmental hypoxia exposure.

BACKGROUND: Formula-fed infants are at increased risk of infections. Due to the cross-talk between the mucosal systems of the gastrointestinal and respiratory tracts, adding synbiotics (prebiotics and probiotics) to infant formula may prevent infections even at distant sites. Infants that were born full term and weaned from breast milk were randomized to prebiotic formula (fructo- and galactooligosaccharides) or the same prebiotic formula with Lactobacillus paracasei ssp. paracasei F19 (synbiotics) from 1 to 6 months of age. The objective was to examine the synbiotic effects on gut microbiota development. RESULTS: Fecal samples collected at ages 1, 4, 6, and 12 months were analyzed using 16S rRNA gene sequencing and a combination of untargeted gas chromatography-mass spectrometry/liquid chromatography-mass spectrometry. These analyses revealed that the synbiotic group had a lower abundance of Klebsiella, a higher abundance of Bifidobacterium breve compared to the prebiotic group, and increases in the anti-microbial metabolite d-3-phenyllactic acid. We also analyzed the fecal metagenome and antibiotic resistome in the 11 infants that had been diagnosed with lower respiratory tract infection (cases) and 11 matched controls using deep metagenomic sequencing. Cases with lower respiratory tract infection had a higher abundance of Klebsiella species and antimicrobial resistance genes related to Klebsiella pneumoniae, compared to controls. The results obtained using 16S rRNA gene amplicon and metagenomic sequencing were confirmed in silico by successful recovery of the metagenome-assembled genomes of the bacteria of interest. CONCLUSIONS: This study demonstrates the additional benefit of feeding specific synbiotics to formula-fed infants over prebiotics only. Synbiotic feeding led to the underrepresentation of Klebsiella, enrichment of bifidobacteria, and increases in microbial degradation metabolites implicated in immune signaling and in the gut-lung and gut-skin axes. Our findings support future clinical evaluation of synbiotic formula in the prevention of infections and associated antibiotic treatment as a primary outcome when breastfeeding is not feasible. TRIAL REGISTRATION: ClinicalTrials.gov NCT01625273 . Retrospectively registered on 21 June 2012.

BACKGROUND: Although the extreme environmental adaptation of organisms is a hot topic in evolutionary biology, genetic adaptation to high-altitude environment remains poorly characterized in ectothermic animals. Squamates are among the most diverse terrestrial vertebrates, with tremendous ecological plasticity and karyotype diversity, and are a unique model system to investigate the genetic footprints of adaptation. RESULTS: We report the first chromosome-level assembly of the Mongolian racerunner (Eremias argus) and our comparative genomics analyses found that multiple chromosome fissions/fusions events are unique to lizards. We further sequenced the genomes of 61 Mongolian racerunner individuals that were collected from altitudes ranging from ~ 80 to ~ 2600 m above sea level (m.a.s.l.). Population genomic analyses revealed many novel genomic regions under strong selective sweeps in populations endemic to high altitudes. Genes embedded in those genomic regions are mainly associated with energy metabolism and DNA damage repair pathways. Moreover, we identified and validated two substitutions of PHF14 that may enhance the lizards' tolerance to hypoxia at high altitudes. CONCLUSIONS: Our study reveals the molecular mechanism of high-altitude adaptation in ectothermic animal using lizard as a research subject and provides a high-quality lizard genomic resource for future research.

BACKGROUND: Amaranthus L. is a diverse genus consisting of domesticated, weedy, and non-invasive species distributed around the world. Nine species are dioecious, of which Amaranthus palmeri S. Watson and Amaranthus tuberculatus (Moq.) J.D. Sauer are troublesome weeds of agronomic crops in the USA and elsewhere. Shallow relationships among the dioecious Amaranthus species and the conservation of candidate genes within previously identified A. palmeri and A. tuberculatus male-specific regions of the Y (MSYs) in other dioecious species are poorly understood. In this study, seven genomes of dioecious amaranths were obtained by paired-end short-read sequencing and combined with short reads of seventeen species in the family Amaranthaceae from NCBI database. The species were phylogenomically analyzed to understand their relatedness. Genome characteristics for the dioecious species were evaluated and coverage analysis was used to investigate the conservation of sequences within the MSY regions. RESULTS: We provide genome size, heterozygosity, and ploidy level inference for seven newly sequenced dioecious Amaranthus species and two additional dioecious species from the NCBI database. We report a pattern of transposable element proliferation in the species, in which seven species had more Ty3 elements than copia elements while A. palmeri and A. watsonii had more copia elements than Ty3 elements, similar to the TE pattern in some monoecious amaranths. Using a Mash-based phylogenomic analysis, we accurately recovered taxonomic relationships among the dioecious Amaranthus species that were previously identified based on comparative morphology. Coverage analysis revealed eleven candidate gene models within the A. palmeri MSY region with male-enriched coverages, as well as regions on scaffold 19 with female-enriched coverage, based on A. watsonii read alignments. A previously reported FLOWERING LOCUS T (FT) within A. tuberculatus MSY contig was also found to exhibit male-enriched coverages for three species closely related to A. tuberculatus but not for A. watsonii reads. Additional characterization of the A. palmeri MSY region revealed that 78% of the region is made of repetitive elements, typical of a sex determination region with reduced recombination. CONCLUSIONS: The results of this study further increase our understanding of the relationships among the dioecious species of the Amaranthus genus as well as revealed genes with potential roles in sex function in the species.

BACKGROUND: Climate change is one of the main factors shaping the distribution and biodiversity of organisms, among others by greatly altering water availability, thus exposing species and ecosystems to harsh desiccation conditions. However, most of the studies so far have focused on the effects of increased temperature. Integrating transcriptomics and physiology is key to advancing our knowledge on how species cope with desiccation stress, and these studies are still best accomplished in model organisms. RESULTS: Here, we characterized the natural variation of European D. melanogaster populations across climate zones and found that strains from arid regions were similar or more tolerant to desiccation compared with strains from temperate regions. Tolerant and sensitive strains differed not only in their transcriptomic response to stress but also in their basal expression levels. We further showed that gene expression changes in tolerant strains correlated with their physiological response to desiccation stress and with their cuticular hydrocarbon composition, and functionally validated three of the candidate genes identified. Transposable elements, which are known to influence stress response across organisms, were not found to be enriched nearby differentially expressed genes. Finally, we identified several tRNA-derived small RNA fragments that differentially targeted genes in response to desiccation stress. CONCLUSIONS: Overall, our results showed that basal gene expression differences across individuals should be analyzed if we are to understand the genetic basis of differential stress survival. Moreover, tRNA-derived small RNA fragments appear to be relevant across stress responses and allow for the identification of stress-response genes not detected at the transcriptional level.

BACKGROUND: Cellular entry of SARS-CoV-2 has been shown to rely on angiotensin-converting enzyme 2 (ACE2) receptors, whose expression in the testis is among the highest in the body. Additionally, the risk of mortality seems higher among male COVID-19 patients, and though much has been published since the first cases of COVID-19, there remain unanswered questions regarding SARS-CoV-2 impact on testes and potential consequences for reproductive health. We investigated testicular alterations in non-vaccinated deceased COVID-19-patients, the precise location of the virus, its replicative activity, and the immune, vascular, and molecular fluctuations involved in the pathogenesis. RESULTS: We found that SARS-CoV-2 testicular tropism is higher than previously thought and that reliable viral detection in the testis requires sensitive nanosensors or RT-qPCR using a specific methodology. Through an in vitro experiment exposing VERO cells to testicular macerates, we observed viral content in all samples, and the subgenomic RNA's presence reinforced the replicative activity of SARS-CoV-2 in testes of the severe COVID-19 patients. The cellular structures and viral particles, observed by transmission electron microscopy, indicated that macrophages and spermatogonial cells are the main SARS-CoV-2 lodging sites, where new virions form inside the endoplasmic reticulum Golgi intermediate complex. Moreover, we showed infiltrative infected monocytes migrating into the testicular parenchyma. SARS-CoV-2 maintains its replicative and infective abilities long after the patient's infection. Further, we demonstrated high levels of angiotensin II and activated immune cells in the testes of deceased patients. The infected testes show thickening of the tunica propria, germ cell apoptosis, Sertoli cell barrier loss, evident hemorrhage, angiogenesis, Leydig cell inhibition, inflammation, and fibrosis. CONCLUSIONS: Our findings indicate that high angiotensin II levels and activation of mast cells and macrophages may be critical for testicular pathogenesis. Importantly, our findings suggest that patients who become critically ill may exhibit severe alterations and harbor the active virus in the testes.

BACKGROUND: Pruning that selectively eliminates unnecessary or incorrect neurites is required for proper wiring of the mature nervous system. During Drosophila metamorphosis, dendritic arbourization sensory neurons (ddaCs) and mushroom body (MB) γ neurons can selectively prune their larval dendrites and/or axons in response to the steroid hormone ecdysone. An ecdysone-induced transcriptional cascade plays a key role in initiating neuronal pruning. However, how downstream components of ecdysone signalling are induced remains not entirely understood. RESULTS: Here, we identify that Scm, a component of Polycomb group (PcG) complexes, is required for dendrite pruning of ddaC neurons. We show that two PcG complexes, PRC1 and PRC2, are important for dendrite pruning. Interestingly, depletion of PRC1 strongly enhances ectopic expression of Abdominal B (Abd-B) and Sex combs reduced, whereas loss of PRC2 causes mild upregulation of Ultrabithorax and Abdominal A in ddaC neurons. Among these Hox genes, overexpression of Abd-B causes the most severe pruning defects, suggesting its dominant effect. Knockdown of the core PRC1 component Polyhomeotic (Ph) or Abd-B overexpression selectively downregulates Mical expression, thereby inhibiting ecdysone signalling. Finally, Ph is also required for axon pruning and Abd-B silencing in MB γ neurons, indicating a conserved function of PRC1 in two types of pruning. CONCLUSIONS: This study demonstrates important roles of PcG and Hox genes in regulating ecdysone signalling and neuronal pruning in Drosophila. Moreover, our findings suggest a non-canonical and PRC2-independent role of PRC1 in Hox gene silencing during neuronal pruning.

BACKGROUND: Sex determination occurs across animal species, but most of our knowledge about its mechanisms comes from only a handful of bilaterian taxa. This limits our ability to infer the evolutionary history of sex determination within animals. RESULTS: In this study, we generated a linkage map of the genome of the colonial cnidarian Hydractinia symbiolongicarpus and used it to demonstrate that this species has an XX/XY sex determination system. We demonstrate that the X and Y chromosomes have pseudoautosomal and non-recombining regions. We then use the linkage map and a method based on the depth of sequencing coverage to identify genes encoded in the non-recombining region and show that many of them have male gonad-specific expression. In addition, we demonstrate that recombination rates are enhanced in the female genome and that the haploid chromosome number in Hydractinia is n = 15. CONCLUSIONS: These findings establish Hydractinia as a tractable non-bilaterian model system for the study of sex determination and the evolution of sex chromosomes.

BACKGROUND: Aging is known to exert an effect on liver regeneration, with the ability of liver to regenerate displaying a significant decline over time. Liver physiological parameters such as liver volume, blood flow, and metabolism, as well as the ability to regenerate after injury have all been shown to decrease at old age in humans and model systems, with a number of molecular mechanisms proposed to be involved, including DNA methylation-dependent genome remodeling. To address how changes in DNA methylation mediate the adverse aging effect on liver regeneration, we searched for differentially methylated genomic regions (DMRs) in mouse livers co-regulated by aging and regeneration and determined their associated genes and enriched pathways. RESULTS: DMRs were identified using whole-genome bisulfite sequencing (WGBS). Pathway analysis of aging DMR-mapped genes revealed two distinct phases of aging, 2-to-8 and 8-to-16 months old (m/o). Regenerative DMR-mapped differentially expressed genes (DEGs) were enriched in pathways controlling cell proliferation and differentiation. Most DMRs shared by both aging and regeneration changed in the same methylation direction between 2 and 8 m/o but in the opposite direction between 8 and 16 m/o. Regenerative DMRs inversely affected by aging during 8-to-16 m/o were found in the promoter/gene regions of 12 genes. Four regenerative DEGs were synchronously regulated by early aging and inversely regulated by mid-to-late aging DMRs. Lead DMR-mapped genes were validated by their expression profiles in liver aging and regeneration. CONCLUSIONS: Our study has uncovered new DMRs and gene targets inversely affected by liver aging and regeneration to explain the adverse aging effect on liver regeneration. These findings will be of fundamental importance to understand the epigenomic changes underlying the biology of aging on liver regeneration.

BACKGROUND: Intracellular hemoglobin polymerization has been supposed to be the major determinant for the elevated rigidity/stiffness of sickle erythrocytes from sickle cell anemia (SCA) patients. However, the contribution of the cell envelope remains unclear. RESULTS: In this study, using atomic force microscopy (AFM), we compared the normal and sickled erythrocyte surfaces for stiffness and topography. AFM detected that sickle cells had a rougher surface and were stiffer than normal erythrocytes and that sickle cell ghosts had a rougher surface (for both outer and inner surfaces) and were thicker than normal ghosts, the latter implying a higher membrane-associated hemoglobin content/layer in the sickle cell envelope. Compared to healthy subjects, the SCA patients had lower plasma lipoprotein levels. AFM further revealed that a mild concentration of methyl-β-cyclodextrin (MβCD, a putative cholesterol-depleting reagent) could induce an increase in roughness of erythrocytes/ghosts and a decrease in thickness of ghosts for both normal and sickle cells, implying that MβCD can alter the cell envelope from outside (cholesterol in the plasma membrane) to inside (membrane-associated hemoglobin). More importantly, MβCD also caused a more significant decrease in stiffness of sickle cells than that of normal erythrocytes. CONCLUSIONS: The data reveal that besides the cytosolic hemoglobin fibers, the cell envelope containing the membrane-associated hemoglobin also is involved in the biomechanical properties (e.g., stiffness and shape maintenance) of sickle erythrocytes.