CRISPR-dCas9 based DNA detection scheme for diagnostics in resource-limited settings.

Nucleic-acid detection is crucial for basic research as well as for applications in medicine such as diagnostics. In resource-limited settings, however, most DNA-detection diagnostic schemes are inapplicable since they rely on expensive machinery, electricity, and trained personnel. Here, we present an isothermal DNA detection scheme for the diagnosis of pathogenic DNA in resource-limited settings. DNA was extracted from urine and blood samples using two different instrument-free methods, and amplified using Recombinase Polymerase Amplification with a sensitivity of <10 copies of DNA within 15 minutes. Target DNA was bound by dCas9/sgRNA that was labelled with a DNA oligomer to subsequently induce Rolling Circle Amplification. This second amplification step produced many copies of a G-quadruplex DNA structure that facilitates a colorimetric readout that is visible to the naked eye. This isothermal DNA-detection scheme can be performed at temperatures between 20-45 °C. As an example of the applicability of the approach, we isothermally (23 °C) detected DNA from a parasite causing visceral leishmaniasis that was spiked into buffer and resulted in a sensitivity of at least 1 zeptomole. For proof of principle, DNA spiked into blood was coupled to the CRISPR-dCas9-based detection scheme yielding a colorimetric readout visible to the naked eye. Given the versatility of the guide-RNA programmability of targets, we envision that this DNA detection scheme can be adapted to detect any DNA with minimal means, which facilitates applications such as point-of-care diagnostics in resource-limited settings.

Diagnosing point-of-care diagnostics for neglected tropical diseases.

Inadequate and nonintegrated diagnostics are the Achilles' heel of global efforts to monitor, control, and eradicate neglected tropical diseases (NTDs). While treatment is often available, NTDs are endemic among marginalized populations, due to the unavailability or inadequacy of diagnostic tests that cause empirical misdiagnoses. The need of the hour is early diagnosis at the point-of-care (PoC) of NTD patients. Here, we review the status quo of PoC diagnostic tests and practices for all of the 24 NTDs identified in the World Health Organization's (WHO) 2021-2030 roadmap, based on their different diagnostic requirements. We discuss the capabilities and shortcomings of current diagnostic tests, identify diagnostic needs, and formulate prerequisites of relevant PoC tests. Next to technical requirements, we stress the importance of availability and awareness programs for establishing PoC tests that fit endemic resource-limited settings. Better understanding of NTD diagnostics will pave the path for setting realistic goals for healthcare in areas with minimal resources, thereby alleviating the global healthcare burden.

αv-Class integrin binding to fibronectin is solely mediated by RGD and unaffected by an RGE mutation.

Fibronectin (FN) is an essential glycoprotein of the extracellular matrix; binds integrins, syndecans, collagens, and growth factors; and is assembled by cells into complex fibrillar networks. The RGD motif in FN facilitates cell binding- and fibrillogenesis through binding to α5ß1 and αv-class integrins. However, whether RGD is the sole binding site for αv-class integrins is unclear. Most notably, substituting aspartate with glutamate (RGE) was shown to eliminate integrin binding in vitro, while mouse genetics revealed that FNRGE preserves αv-class integrin binding and fibrillogenesis. To address this conflict, we employed single-cell force spectroscopy, engineered cells, and RGD motif-deficient mice (Fn1ΔRGD/ΔRGD) to search for additional αv-class integrin-binding sites. Our results demonstrate that α5ß1 and αv-class integrins solely recognize the FN-RGD motif and that αv-class, but not α5ß1, integrins retain FN-RGE binding. Furthermore, Fn1ΔRGD/ΔRGD tissues and cells assemble abnormal and dysfunctional FNΔRGD fibrils in a syndecan-dependent manner. Our data highlight the central role of FN-RGD and the functionality of FN-RGE for αv-class integrins.

Matching Development of Point-of-Care Diagnostic Tests to the Local Context: A Case Study of Visceral Leishmaniasis in Kenya and Uganda.

The rapid growth of point-of-care (POC) diagnostic tests necessitates a clear vision of when, where, and why a new POC diagnostic test needs to be developed and how it can be used in a way that matches a local health care context. Here, we present an innovative approach toward developing a concept target product profile (CTPP), which is a new mapping tool that helps researchers match a new diagnostic test to a specific local health care context early in the research and development process. As a case study, we focus on the diagnosis of visceral leishmaniasis (VL) in rural resource-limited regions of Kenya and Uganda. Our stepwise approach integrates elements of design thinking and uses a combination of literature reviews and field research for a context analysis of local health care systems and practices. We then use visual thinking in the form of Gigamaps and patient journeys to identify use case scenarios and to present our findings from the field research to key stakeholders. The use case scenarios describe the diagnostic scope of a new POC test based on the feasibility of the new test, the local need, and the contextual fit. For our case study of VL, we identify 2 valuable use case scenarios, namely test-of-cure and screening and confirmation, and we formulate a CTPP. We anticipate that a CTPP will enable researchers to match a new POC diagnostic test during the research and development process to the local health care context in which it will be used.

Corrigendum: Fibronectin-bound α5ß1 integrins sense load and signal to reinforce adhesion in less than a second.

This corrects the article DOI: 10.1038/nmat5023.

Fibronectin-bound α5ß1 integrins sense load and signal to reinforce adhesion in less than a second.

Integrin-mediated mechanosensing of the extracellular environment allows cells to control adhesion and signalling. Whether cells sense and respond to force immediately upon ligand-binding is unknown. Here, we report that during adhesion initiation, fibroblasts respond to mechanical load by strengthening integrin-mediated adhesion to fibronectin (FN) in a biphasic manner. In the first phase, which depends on talin and kindlin as well as on the actin nucleators Arp2/3 and mDia, FN-engaged α5ß1 integrins activate focal adhesion kinase (FAK) and c-Src in less than 0.5 s to steeply strengthen α5ß1- and αV-class integrin-mediated adhesion. When the mechanical load exceeds a certain threshold, fibroblasts decrease adhesion and initiate the second phase, which is characterized by less steep adhesion strengthening. This unique, biphasic cellular adhesion response is mediated by α5ß1 integrins, which form catch bonds with FN and signal to FN-binding integrins to reinforce cell adhesion much before visible adhesion clusters are formed.

αV-class integrins exert dual roles on α5ß1 integrins to strengthen adhesion to fibronectin.

Upon binding to the extracellular matrix protein, fibronectin, αV-class and α5ß1 integrins trigger the recruitment of large protein assemblies and strengthen cell adhesion. Both integrin classes have been functionally specified, however their specific roles in immediate phases of cell attachment remain uncharacterized. Here, we quantify the adhesion of αV-class and/or α5ß1 integrins expressing fibroblasts initiating attachment to fibronectin (≤120 s) by single-cell force spectroscopy. Our data reveals that αV-class integrins outcompete α5ß1 integrins. Once engaged, αV-class integrins signal to α5ß1 integrins to establish additional adhesion sites to fibronectin, away from those formed by αV-class integrins. This crosstalk, which strengthens cell adhesion, induces α5ß1 integrin clustering by RhoA/ROCK/myosin-II and Arp2/3-mediated signalling, whereas overall cell adhesion depends on formins. The dual role of both fibronectin-binding integrin classes commencing with an initial competition followed by a cooperative crosstalk appears to be a basic cellular mechanism in assembling focal adhesions to the extracellular matrix.

The fibronectin synergy site re-enforces cell adhesion and mediates a crosstalk between integrin classes.

Fibronectin (FN), a major extracellular matrix component, enables integrin-mediated cell adhesion via binding of α5ß1, αIIbß3 and αv-class integrins to an RGD-motif. An additional linkage for α5 and αIIb is the synergy site located in close proximity to the RGD motif. We report that mice with a dysfunctional FN-synergy motif (Fn1syn/syn) suffer from surprisingly mild platelet adhesion and bleeding defects due to delayed thrombus formation after vessel injury. Additional loss of ß3 integrins dramatically aggravates the bleedings and severely compromises smooth muscle cell coverage of the vasculature leading to embryonic lethality. Cell-based studies revealed that the synergy site is dispensable for the initial contact of α5ß1 with the RGD, but essential to re-enforce the binding of α5ß1/αIIbß3 to FN. Our findings demonstrate a critical role for the FN synergy site when external forces exceed a certain threshold or when αvß3 integrin levels decrease below a critical level.

Kindlin-2 cooperates with talin to activate integrins and induces cell spreading by directly binding paxillin.

Integrins require an activation step prior to ligand binding and signaling. How talin and kindlin contribute to these events in non-hematopoietic cells is poorly understood. Here we report that fibroblasts lacking either talin or kindlin failed to activate ß1 integrins, adhere to fibronectin (FN) or maintain their integrins in a high affinity conformation induced by Mn(2+). Despite compromised integrin activation and adhesion, Mn(2+) enabled talin- but not kindlin-deficient cells to initiate spreading on FN. This isotropic spreading was induced by the ability of kindlin to directly bind paxillin, which in turn bound focal adhesion kinase (FAK) resulting in FAK activation and the formation of lamellipodia. Our findings show that talin and kindlin cooperatively activate integrins leading to FN binding and adhesion, and that kindlin subsequently assembles an essential signaling node at newly formed adhesion sites in a talin-independent manner.

clpC operon regulates cell architecture and sporulation in Bacillus anthracis.

The clpC operon is known to regulate several processes such as genetic competence, protein degradation and stress survival in bacteria. Here, we describe the role of clpC operon in Bacillus anthracis. We generated knockout strains of the clpC operon genes to investigate the impact of CtsR, McsA, McsB and ClpC deletion on essential processes of B. anthracis. We observed that growth, cell division, sporulation and germination were severely affected in mcsB and clpC deleted strains, while none of deletions affected toxin secretion. Growth defect in these strains was pronounced at elevated temperature. The growth pattern gets restored on complementation of mcsB and clpC in respective mutants. Electron microscopic examination revealed that mcsB and clpC deletion also causes defect in septum formation leading to cell elongation. These vegetative cell deformities were accompanied by inability of mutant strains to generate morphologically intact spores. Higher levels of polyhydroxybutyrate granules accumulation were also observed in these deletion strains, indicating a defect in sporulation process. Our results demonstrate, for the first time, the vital role played by McsB and ClpC in physiology of B. anthracis and open up further interest on this operon, which might be of importance to success of B. anthracis as pathogen.

Assay for characterizing the recovery of vertebrate cells for adhesion measurements by single-cell force spectroscopy.

Single-cell force spectroscopy (SCFS) is becoming a widely used method to quantify the adhesion of a living cell to a substrate, another cell or tissue. The high sensitivity of SCFS permits determining the contributions of individual cell adhesion molecules (CAMs) to the adhesion force of an entire cell. However, to prepare adherent cells for SCFS, they must first be detached from tissue-culture flasks or plates. EDTA and trypsin are often applied for this purpose. Because cellular properties can be affected by this treatment, cells need to recover before being further characterized by SCFS. Here we introduce atomic force microscopy (AFM)-based SCFS to measure the mechanical and adhesive properties of HeLa cells and mouse embryonic kidney fibroblasts while they are recovering after detachment from tissue-culture. We find that mechanical and adhesive properties of both cell lines recover quickly (<10 min) after detachment using EDTA, while trypsin-detached fibroblasts require >60 min to fully recover. Our assay introduced to characterize the recovery of mammalian cells after detachment can in future be used to estimate the recovery behavior of other adherent cell types.

A practical guide to quantify cell adhesion using single-cell force spectroscopy.

Quantitative analysis of cellular interactions with the extracellular environment is necessary to gain an understanding of how cells regulate adhesion in the development and maintenance of multicellular organisms, and how changes in cell adhesion contribute to diseases. We provide a practical guide to quantify the adhesive strength of living animal cells to various substrates using atomic force microscopy (AFM)-based single-cell force spectroscopy (SCFS). We describe how to control cell state and attachment to the AFM cantilever, how to functionalize supports for SCFS measurements, how to conduct cell adhesion measurements, and how to analyze and interpret the recorded SCFS data. This guide is intended to assist newcomers in the field to perform AFM-based SCFS measurements.

Plasmodium falciparum reticulocyte binding-like homologue protein 2 (PfRH2) is a key adhesive molecule involved in erythrocyte invasion.

Erythrocyte invasion by Plasmodium merozoites is a complex, multistep process that is mediated by a number of parasite ligand-erythrocyte receptor interactions. One such family of parasite ligands includes the P. falciparum reticulocyte binding homologue (PfRH) proteins that are homologous with the P. vivax reticulocyte binding proteins and have been shown to play a role in erythrocyte invasion. There are five functional PfRH proteins of which only PfRH2a/2b have not yet been demonstrated to bind erythrocytes. In this study, we demonstrated that native PfRH2a/2b is processed near the N-terminus yielding fragments of 220 kDa and 80 kDa that exhibit differential erythrocyte binding specificities. The erythrocyte binding specificity of the 220 kDa processed fragment of native PfRH2a/2b was sialic acid-independent, trypsin resistant and chymotrypsin sensitive. This specific binding phenotype is consistent with previous studies that disrupted the PfRH2a/2b genes and demonstrated that PfRH2b is involved in a sialic acid independent, trypsin resistant, chymotrypsin sensitive invasion pathway. Interestingly, we found that the smaller 80 kDa PfRH2a/2b fragment is processed from the larger 220 kDa fragment and binds erythrocytes in a sialic acid dependent, trypsin resistant and chymotrypsin sensitive manner. Thus, the two processed fragments of PfRH2a/2b differed with respect to their dependence on sialic acids for erythrocyte binding. Further, we mapped the erythrocyte binding domain of PfRH2a/2b to a conserved 40 kDa N-terminal region (rPfRH2(40)) in the ectodomain that is common to both PfRH2a and PfRH2b. We demonstrated that recombinant rPfRH2(40) bound human erythrocytes with the same specificity as the native 220 kDa processed protein. Moreover, antibodies generated against rPfRH2(40) blocked erythrocyte invasion by P. falciparum through a sialic acid independent pathway. PfRH2a/2b thus plays a key role in erythrocyte invasion and its conserved receptor-binding domain deserves attention as a promising candidate for inclusion in a blood-stage malaria vaccine.