Overburden in Caregivers of Chronically Ill Adolescents: A Comparative Study between Pulmonological and Endocrinological Diseases.

Chronic pediatric disease has a major impact on the life of a child and his or her family. In this sense, the figure of the main caregiver is relevant, who may present adjustment difficulties before the disease, accompanied by high levels of stress and emotional discomfort, which interfere with the adolescent's adjustment before the new situation. The aim of this research was to carry out a comparative study among caregivers of adolescents with various diseases, analyzing the risk and protective factors for the stress presented by this population. For this purpose, a sample of 406 main caregivers of adolescents with an endocrinological or pneumological disease from different hospitals in Valencia was used, where characteristics related to the disease, attachment, type of family, and emotional distress of these caregivers were considered. In general, the results show higher levels of stress in caregivers of adolescents with an endocrinological disease, specifically DM1, and found significant predictors of anxiety-depressive symptomatology, the need for approval, and several variables related to the disease. These data reveal the importance of providing comprehensive care to the family system, offering health skills to overcome diseases, and reinforcing the protective factors offered by the family system.

Colorectal Cancer Stem Cells Fuse with Monocytes to Form Tumour Hybrid Cells with the Ability to Migrate and Evade the Immune System.

BACKGROUND: The cancer cell fusion theory could be one of the best explanations for the metastasis from primary tumours. METHODS: Herein, we co-cultured colorectal cancer (CRC) stem cells with human monocytes and analysed the properties of the generated tumour hybrid cells (THCs). The presence of THCs in the bloodstream together with samples from primary and metastatic lesions and their clinical correlations were evaluated in CRC patients and were detected by both FACS and immunofluorescence methods. Additionally, the role of SIGLEC5 as an immune evasion molecule in colorectal cancer was evaluated. RESULTS: Our data demonstrated the generation of THCs after the in vitro co-culture of CRC stem cells and monocytes. These cells, defined as CD45+CD14+EpCAM+, showed enhanced migratory and proliferative abilities. The THC-specific cell surface signature allows identification in matched primary tumour tissues and metastases as well as in the bloodstream from patients with CRC, thus functioning as a biomarker. Moreover, SIG-LEC5 expression on in vitro generated THCs has shown to be involved in the mechanism for immune evasion. Additionally, sSIGLEC5 levels correlated with THC numbers in the prospective cohort of patients. CONCLUSIONS: Our results indicate the generation of a hybrid entity after the in vitro co-culture between CRC stem cells and human monocytes. Moreover, THC numbers present in patients are related to both prognosis and the later spread of metastases in CRC patients.

Intertwined leukocyte balances in tumours and peripheral blood as robust predictors of right and left colorectal cancer survival.

BACKGROUND: Colorectal cancer (CRC) accounts for 9.4% of overall cancer deaths, ranking second after lung cancer. Despite the large number of factors tested to predict their outcome, most patients with similar variables show big differences in survival. Moreover, right-sided CRC (RCRC) and left-sided CRC (LCRC) patients exhibit large differences in outcome after surgical intervention as assessed by preoperative blood leukocyte status. We hypothesised that stronger indexes than circulating (blood) leukocyte ratios to predict RCRC and LCRC patient outcomes will result from combining both circulating and infiltrated (tumour/peritumour fixed tissues) concentrations of leukocytes. AIM: To seek variables involving leukocyte balances in peripheral blood and tumour tissues and to predict the outcome of CRC patients. METHODS: Sixty-five patients diagnosed with colon adenocarcinoma by the Digestive Surgery Service of the La Paz University Hospital (Madrid, Spain) were enrolled in this study: 43 with RCRC and 22 with LCRC. Patients were followed-up from January 2017 to March 2021 to record overall survival (OS) and recurrence-free survival (RFS) after surgical interventions. Leukocyte concentrations in peripheral blood were determined by routine laboratory protocols. Paraffin-fixed samples of tumour and peritumoural tissues were assessed for leukocyte concentrations by immunohistochemical detection of CD4, CD8, and CD14 marker expression. Ratios of leukocyte concentration in blood and tissues were calculated and evaluated for their predictor values for OS and RFS with Spearman correlations and Cox univariate and multivariate proportional hazards regression, followed by the calculation of the receiver-operating characteristic and area under the curve (AUC) and the determination of Youden's optimal cutoff values for those variables that significantly correlated with either RCRC or LCRC patient outcomes. RCRC patients from the cohort were randomly assigned to modelling and validation sets, and clinician-friendly nomograms were developed to predict OS and RFS from the respective significant indexes. The accuracy of the model was evaluated using calibration and validation plots. RESULTS: The relationship of leukocyte ratios in blood and peritumour resulted in six robust predictors of worse OS in RCRC: CD8+ lymphocyte content in peritumour (CD8pt, AUC = 0.585, cutoff < 8.250, P = 0.0077); total lymphocyte content in peritumour (CD4CD8pt, AUC = 0.550, cutoff < 10.160, P = 0.0188); lymphocyte-to-monocyte ratio in peritumour (LMRpt, AUC = 0.807, cutoff < 3.185, P = 0.0028); CD8+ LMR in peritumour (CD8MRpt, AUC = 0.757, cutoff < 1.650, P = 0.0007); the ratio of blood LMR to LMR in peritumour (LMRb/LMRpt, AUC = 0.672, cutoff > 0.985, P = 0.0244); and the ratio of blood LMR to CD8+ LMR in peritumour (LMRb/CD8MRpt, AUC = 0.601, cutoff > 1.485, P = 0.0101). In addition, three robust predictors of worse RFS in RCRC were found: LMRpt (AUC = 0.737, cutoff < 3.185, P = 0.0046); LMRb/LMRpt (AUC = 0.678, cutoff > 0.985, P = 0.0155) and LMRb/CD8MRpt (AUC = 0.615, cutoff > 1.485, P = 0.0141). Furthermore, the ratio of blood LMR to CD4+ LMR in peritumour (LMRb/CD4MRpt, AUC = 0.786, cutoff > 10.570, P = 0.0416) was found to robustly predict poorer OS in LCRC patients. The nomograms showed moderate accuracy in predicting OS and RFS in RCRC patients, with concordance index of 0.600 and 0.605, respectively. CONCLUSION: Easily obtainable variables at preoperative consultation, defining the status of leukocyte balances between peripheral blood and peritumoural tissues, are robust predictors for OS and RFS of both RCRC and LCRC patients.

The fimbrial usher FimD follows the SurA-BamB pathway for its assembly in the outer membrane of Escherichia coli.

Fimbrial ushers are the largest ß-barrel outer membrane proteins (OMPs) known to date, which function in the polymerization of fimbriae and their translocation to the bacterial surface. Folding and assembly of these complex OMPs are not characterized. Here, we investigate the role of periplasmic chaperones (SurA, Skp, DegP, and FkpA) and individual components of the ß-barrel assembly machinery (BAM) complex (BamA, BamB, BamC, and BamE) in the folding of the Escherichia coli FimD usher. The FimD level is dramatically reduced (∼30-fold) in a surA null mutant, but a strong cell envelope stress is constitutively activated with upregulation of DegP (∼10-fold). To demonstrate a direct role of SurA, FimD folding was analyzed in a conditional surA mutant in which SurA expression was controlled. In this strain, FimD is depleted from bacteria in parallel to SurA without significant upregulation of DegP. Interestingly, the dependency on SurA is higher for FimD than for other OMPs. We also demonstrate that a functional BAM complex is needed for folding of FimD. In addition, FimD levels were strongly reduced (∼5-fold) in a mutant lacking the accessory lipoprotein BamB. The critical role of BamB for FimD folding was confirmed by complementation and BamB depletion experiments. Similar to SurA dependency, FimD showed a stronger dependency on BamB than OMPs. On the other hand, folding of FimD was only marginally affected in bamC and bamE mutants. Collectively, our results indicate that FimD usher follows the SurA-BamB pathway for its assembly. The preferential use of this pathway for the folding of OMPs with large ß-barrels is discussed.

Potent phagocytic activity with impaired antigen presentation identifying lipopolysaccharide-tolerant human monocytes: demonstration in isolated monocytes from cystic fibrosis patients.

Monocyte exposure to LPS induces a transient state in which these cells are refractory to further endotoxin stimulation. This phenomenon, termed endotoxin tolerance (ET), is characterized by a decreased production of cytokines in response to the proinflammatory stimulus. We have established a robust model of ET and have determined the time frame and features of LPS unresponsiveness in cultured human monocytes. A large number of genes transcribed in tolerant monocytes were classified as either "tolerizable" or "nontolerizable" depending on their expression levels during the ET phase. Tolerant monocytes exhibit rapid IL-1R-associated kinase-M (IRAK-M) overexpression, high levels of triggering receptor expressed on myeloid cells-1 (TREM-1) and CD64, and a marked down-regulation of MHC molecules and NF-kappaB2. These cells combine potent phagocytic activity with impaired capability for Ag presentation. We also show that circulating monocytes isolated from cystic fibrosis patients share all the determinants that characterize cells locked in an ET state. These findings identify a new mechanism that contributes to impaired inflammation in cystic fibrosis patients despite a high frequency of infections. Our results indicate that a tolerant phenotype interferes with timing, efficiency, and outcome of the innate immune responses against bacterial infections.

Comparative analysis of the biochemical and functional properties of C-terminal domains of autotransporters.

Autotransporters (ATs) are the largest group of proteins secreted by Gram-negative bacteria and include many virulence factors from human pathogens. ATs are synthesized as large precursors with a C-terminal domain that is inserted in the outer membrane (OM) and is essential for the translocation of an N-terminal passenger domain to the extracellular milieu. Several mechanisms have been proposed for AT secretion. Self-translocation models suggest transport across a hydrophilic channel formed by an internal pore of the ß-barrel or by the oligomerization of C-terminal domains. Alternatively, an assisted-translocation model suggests that transport employs a conserved machinery of the bacterial OM such as the Bam complex. In this work we have investigated AT secretion by carrying out a comparative study to analyze the conserved biochemical and functional features of different C-terminal domains selected from ATs of gammaproteobacteria, betaproteobacteria, alphaproteobacteria, and epsilonproteobacteria. Our results indicate that C-terminal domains having an N-terminal α-helix and a ß-barrel constitute functional transport units for the translocation of peptides and immunoglobulin domains with disulfide bonds. In vivo and in vitro analyses show that multimerization is not a conserved feature in AT C-terminal domains. Furthermore, we demonstrate that the deletion of the conserved α-helix severely impairs ß-barrel folding and OM insertion and thereby blocks passenger domain secretion. These observations suggest that the AT ß-barrel without its α-helix cannot form a stable hydrophilic channel in the OM for protein translocation. The implications of our data for an understanding of AT secretion are discussed.

Tumor stem cells fuse with monocytes to form highly invasive tumor-hybrid cells.

The 'cancer cell fusion' theory is controversial due to the lack of methods available to identify hybrid cells and to follow the phenomenon in patients. However, it seems to be one of the best explanations for both the origin and metastasis of primary tumors. Herein, we co-cultured lung cancer stem cells with human monocytes and analyzed the dynamics and properties of tumor-hybrid cells (THC), as well as the molecular mechanisms beneath this fusion process by several techniques: electron-microscopy, karyotyping, CRISPR-Cas9, RNA-seq, immunostaining, signaling blockage, among others. Moreover, mice models were assessed for in vivo characterization of hybrids colonization and invasiveness. Then, the presence of THCs in bloodstream and samples from primary and metastatic lesions were detected by FACS and immunofluorescence protocols, and their correlations with TNM stages established. Our data indicate that the generation of THCs depends on the expression of CD36 on tumor stem cells and the oxidative state and polarization of monocytes, the latter being strongly influenced by microenvironmental fluctuations. Highly oxidized M2-like monocytes show the strongest affinity to fuse with tumor stem cells. THCs are able to proliferate, colonize and invade organs. THC-specific cell surface signature CD36+CD14+PANK+ allows identifying them in matched primary tumor tissues and metastases as well as in bloodstream from patients with lung cancer, thus functioning as a biomarker. THCs levels in circulation correlate with TNM classification. Our results suggest that THCs are involved in both origin and spread of metastatic cells. Furthermore, they might set the bases for future therapies to avoid or eradicate lung cancer metastasis.

Role of periplasmic chaperones and BamA (YaeT/Omp85) in folding and secretion of intimin from enteropathogenic Escherichia coli strains.

Intimin is a bacterial adhesin located on the surface of enteropathogenic Escherichia coli and other related bacteria that is believed to self-translocate across the outer membrane (OM), and therefore it has been regarded as a member of the type V secretion system (T5SS), which includes classical autotransporters (ATs). However, intimin has few structural similarities to classical ATs and an opposite topology with an OM-embedded N region and a secreted C region. Since the actual secretion mechanism of intimin is unknown, we investigated intimin biogenesis by analyzing its requirement of periplasmic chaperones (DsbA, SurA, Skp, and DegP) and of OM protein BamA (YaeT/Omp85) for folding, OM insertion, and translocation. Using full-length and truncated intimin polypeptides, we demonstrate that DsbA catalyzes the formation of a disulfide bond in the D3 lectin-like domain of intimin in the periplasm, indicating that this secreted C-terminal domain is at least partially folded prior to its translocation across the OM. We also show that SurA chaperone plays the major role for periplasmic transport and folding of the N region of intimin, whereas the parallel pathway made by Skp and DegP chaperones plays a secondary role in this process. Further, we demonstrate that BamA is essential for the insertion of the N region of intimin in the OM and that the protease activity of DegP participates in the degradation of misfolded intimin. The significance of these findings for a BamA-dependent secretion mechanism of intimin is discussed in the context of T5SSs.

Unraveling Gardnerella vaginalis Surface Proteins Using Cell Shaving Proteomics.

Gardnerella vaginalis is one of the main etiologic agents of bacterial vaginosis (BV). This infection is responsible for a wide range of public health costs and is associated with several adverse outcomes during pregnancy. Improving our understanding of G. vaginalis protein cell surface will assist in BV diagnosis. This study represents the first proteomic approach that has analyzed the exposed proteins on G. vaginalis cell surface using a shaving approach. The 261 G. vaginalis proteins identified using this approach were analyzed with bioinformatic tools to detect characteristic motifs from surface-exposed proteins, such as signal peptides (36 proteins), lipobox domains (17 proteins), LPXTG motifs (5 proteins) and transmembrane alpha-helices (66 proteins). One third of the identified proteins were found to have at least one typical motif of surface-exposed proteins. Furthermore, the subcellular location was examined using two predictors (PSORT and Gpos-mPLoc). These bioinformatic tools classified 17% of the identified proteins as surface-associated proteins. Interestingly, we identified 13 members of the ATP-binding cassette (ABC) superfamily, which were mainly involved in the translocation of various substrates across membranes. To validate the location of the G. vaginalis surface-exposed proteins, an immunofluorescence assay with antibodies against Escherichia coli GroEL was performed to reveal the extracellular location of the moonlighting GroEL. In addition, monoclonal antibodies (mAb) against G. vaginalis Cna protein were produced and used to validate the location of Cna on the surface of the G. vaginalis. These high affinity anti-Cna mAb represent a useful tool for the study of this pathogenic microorganism and the BV.

Nitric oxide induces SOCS-1 expression in human monocytes in a TNF-alpha-dependent manner.

In contrast to the thoroughly characterized mechanisms of positive regulation within cytokine signaling pathways, our knowledge of negative feedback loops is comparatively sparse. We and others have previously reported that IRAK-M down-regulates inflammatory responses to multiple stimuli. In particular, we could show that the nitric oxide (NO) donor, GSNO, induces IRAK-M overexpression in human monocytes. Here we study the expression of another important negative regulator of cytokine signaling, SOCS-1, in human monocytes exposed to GSNO. The NO donor induced significant levels of SOCS-1 mRNA and protein, 6 h and 16 h after stimulation, respectively. Monocytes stimulated with GSNO for longer periods (24 h and 48 h) failed to express IL-6 and IP-10 upon LPS challenge. In addition, and in line with previous reports of NO-mediated induction of TNF-alpha, we have found that exposure to this cytokine induces SOCS-1 mRNA in human monocytes. A blocking antibody against TNF-alpha impaired SOCS-1 expression upon GSNO treatment and re-instated IL-6 and IP-10 mRNA levels after LPS challenge in cultures pretreated with the NO donor. We conclude that NO stimulates SOCS-1 overexpression in a pathway at least partially regulated by TNF-alpha.

High affinity nanobodies against human epidermal growth factor receptor selected on cells by E. coli display.

Most therapeutic antibodies (Abs) target cell surface proteins on tumor and immune cells. Cloning of Ab gene libraries in E. coli and their display on bacteriophages is commonly used to select novel therapeutic Abs binding target antigens, either purified or expressed on cells. However, the sticky nature of bacteriophages renders phage display selections on cells challenging. We previously reported an E. coli display system for expression of VHHs (i.e., nanobodies, Nbs) on the surface of bacteria and selection of high-affinity clones by magnetic cell sorting (MACS). Here, we demonstrate that E. coli display is also an attractive method for isolation of Nbs against cell surface antigens, such as the epidermal growth factor receptor (EGFR), upon direct selection and screening of Ab libraries on live cells. We employ a whole cell-based strategy using a VHH library obtained by immunization with human tumor cells over-expressing EGFR (i.e., A431), and selection of bacterial clones bound to murine fibroblast NIH-3T3 cells transfected with human EGFR, after depletion of non-specific clones on untransfected cells. This strategy resulted in the isolation of high-affinity Nbs binding distinct epitopes of EGFR, including Nbs competing with the ligand, EGF, as characterized by flow cytometry of bacteria displaying the Nbs and binding assays with purified Nbs using surface plasmon resonance. Hence, our study demonstrates that E. coli display of VHH libraries and selection on cells enables efficient isolation and characterization of high-affinity Nbs against cell surface antigens.

Analyzing the Role of Periplasmic Folding Factors in the Biogenesis of OMPs and Members of the Type V Secretion System.

The outer membrane (OM) of gram-negative bacteria is highly packed with OM proteins (OMPs) and the trafficking and assembly of OMPs in gram-negative bacteria is a subject of intense research. Structurally, OMPs vary in the number of ß-strands and in the size and complexity of extra-membrane domains, with extreme examples being the members of the type V protein secretion system (T5SS), such as the autotransporter (AT) and intimin/invasin families of secreted proteins, in which a large extracellular "passenger" domain is linked to a ß-barrel that inserts in the OM. Despite their structural and functional diversity, OMPs interact in the periplasm with a relatively small set of protein chaperones that facilitate their transport from the inner membrane (IM) to the ß-barrel assembly machinery (BAM complex), preventing aggregation and assisting their folding in various aspects including disulfide bond formation. This chapter is focused on the periplasmic folding factors involved in the biogenesis of integral OMPs and members of T5SS in E. coli, which are used as a model system in this field. Background information on these periplasmic folding factors is provided along with genetic methods to generate conditional mutants that deplete these factors from E. coli and biochemical methods to analyze the folding, surface display, disulfide formation and oligomerization state of OMPs/T5SS in these mutants.

Candida albicans Shaving to Profile Human Serum Proteins on Hyphal Surface.

Candida albicans is a human opportunistic fungus and it is responsible for a wide variety of infections, either superficial or systemic. C. albicans is a polymorphic fungus and its ability to switch between yeast and hyphae is essential for its virulence. Once C. albicans obtains access to the human body, the host serum constitutes a complex environment of interaction with C. albicans cell surface in bloodstream. To draw a comprehensive picture of this relevant step in host-pathogen interaction during invasive candidiasis, we have optimized a gel-free shaving proteomic strategy to identify both, human serum proteins coating C. albicans cells and fungi surface proteins simultaneously. This approach was carried out with normal serum (NS) and heat inactivated serum (HIS). We identified 214 human and 372 C. albicans unique proteins. Proteins identified in C. albicans included 147 which were described as located at the cell surface and 52 that were described as immunogenic. Interestingly, among these C. albicans proteins, we identified 23 GPI-anchored proteins, Gpd2 and Pra1, which are involved in complement system evasion and 7 other proteins that are able to attach plasminogen to C. albicans surface (Adh1, Eno1, Fba1, Pgk1, Tdh3, Tef1, and Tsa1). Furthermore, 12 proteins identified at the C. albicans hyphae surface induced with 10% human serum were not detected in other hypha-induced conditions. The most abundant human proteins identified are involved in complement and coagulation pathways. Remarkably, with this strategy, all main proteins belonging to complement cascades were identified on the C. albicans surface. Moreover, we identified immunoglobulins, cytoskeletal proteins, metabolic proteins such as apolipoproteins and others. Additionally, we identified more inhibitors of complement and coagulation pathways, some of them serpin proteins (serine protease inhibitors), in HIS vs. NS. On the other hand, we detected a higher amount of C3 at the C. albicans surface in NS than in HIS, as validated by immunofluorescence.

Percepción y prácticas docentes para una educación inclusiva / Percepção e práticas docentes para uma educação inclusiva / Teacher's perception and practices for an inclusive education

Para que una sociedad evolucionada se construya como inclusiva es imprescindible reconocer, desde la escuela, la diversidad como un aspecto positivo que enriquece los procesos de enseñanza y aprendizaje; y garantizar la igualdad de oportunidades mediante la construcción de entornos seguros, democráticos y equitativos. Para ello, es primordial asegurar actitudes positivas hacia la inclusión entre los profesores. Se presenta una investigación cuyo principal objetivo fue conocer la percepción de docentes de Educación Primaria acerca de la educación inclusiva, así como las estrategias desarrolladas con el fin de favorecerla. Se utilizó una metodología de tipo cuantitativo, no experimental y transversal. Se encuestó a un total de cincuenta y cinco docentes de cinco centros educativos de titularidad pública y concertada de la Región de Murcia (España). Los resultados obtenidos mostraron actitudes positivas hacia la educación inclusiva, si bien los docentes consideraron que su formación inicial era insuficiente para trabajar de manera efectiva la diversidad en las aulas, al igual que los recursos de los que disponen.

For an evolved society to be built as inclusive, it is essential to recognize diversity from school as a positive aspect that enriches teaching and learning processes and guarantees equal opportunities through the construction of safe, democratic and equitable environments. In order to achieve this, it is essential to ensure positive attitudes towards inclusion among teachers. Aresearch is presented in which the main objective was to know the perception of Primary Education teachers regardinginclusive education, as well as the strategies developed in order to favor it. A quantitative, non-experimental and transverse/cross-sectional methodology was used. A total of fifty-five teachers from five publicly-owned and subsidized schools in the Region of Murcia (Spain) were surveyed. The results demonstrated positive attitudes towards inclusive education, although teachers considered that their initial training was insufficient to effectively work with diversity in the classroom, as well as the resources available to them.

Para que uma sociedade evoluída se construa como inclusiva, é imprescindível reconhecer, desde a escola, a diversidade como um aspecto positivo que enriquece os processos de ensino e aprendizagem, assegurando, assim, a igualdade de oportunidades através da construção de ambientes seguros, democráticos e equitativos. Para isso, é essencial garantir, entre os professores, atitudes positivas em relação à inclusão. Essa pesquisa tem como objetivo conhecer a percepção dos professores do ensino fundamental sobre a educação inclusiva, assim como as estratégias desenvolvidas para favorecê-la. Foi utilizada uma metodologia de tipo quantitativa, não experimental e transversal. Cinquenta e cinco professores de cinco instituições educativas públicas da região de Murcia (Espanha) foram entrevistados. Os resultados obtidos mostraram atitudes positivas em relação à educação inclusiva, embora os professores considerassem que sua formação inicial era insuficiente para trabalhar efetivamente a diversidade nas salas de aula, bem como consideraram insuficientes os recursos disponíveis.

Selection of single domain antibodies from immune libraries displayed on the surface of E. coli cells with two ß-domains of opposite topologies.

Screening of antibody (Ab) libraries by direct display on the surface of E. coli cells is hampered by the presence of the outer membrane (OM). In this work we demonstrate that the native ß-domains of EhaA autotransporter and intimin, two proteins from enterohemorrhagic E. coli O157:H7 (EHEC) with opposite topologies in the OM, are effective systems for the display of immune libraries of single domain Abs (sdAbs) from camelids (nanobodies or VHH) on the surface of E. coli K-12 cells and for the selection of high affinity sdAbs using magnetic cell sorting (MACS). We analyzed the capacity of EhaA and intimin ß-domains to display individual sdAbs and sdAb libraries obtained after immunization with the extracellular domain of the translocated intimin receptor from EHEC (TirM(EHEC)). We demonstrated that both systems displayed functional sdAbs on the surface of E. coli cells with little proteolysis and cellular toxicity, although E. coli cells displaying sdAbs with the ß-domain of intimin showed higher antigen-binding capacity. Both E. coli display libraries were screened for TirM(EHEC) binding clones by MACS. High affinity binders were selected by both display systems, although more efficiently with the intimin ß-domain. The specificity of the selected clones against TirM(EHEC) was demonstrated by flow cytometry of E. coli cells, along with ELISA and surface plasmon resonance with purified sdAbs. Finally, we employed the E. coli cell display systems to provide an estimation of the affinity of the selected sdAb by flow cytometry analysis under equilibrium conditions.