Electrical bioimpedance analysis and comparison in biological tissues through crystalloid solutions implementation.

Electrical bioimpedance is a non-invasive and radiation-free technique that was proposed to be used in different clinical areas, however, its practical use is limited due to its low capacity to discriminate between tissues. In order to overcome this limitation, our research group proposes to incorporate the contrast media into the electrical bioimpedance procedure. The main objective of the present study was to assess the crystalloid solutions as a possible contrast media to discriminate between different tissue types in the bioimpedance technique. Two medical-grade crystalloid solutions (Hartmann and NaCl 0.9%) were injected into three biological ex vivo models: kidney, liver, and brain. BIOPAC system was used to acquire bioimpedance data before and after the injections. The data was adjusted to the Debye electrical model. The analysis of measured values showed substantial bioimpedance disparities in tissues subjected to isotonic solutions. The NaCl solution exhibited more pronounced changes in electrical parameters compared to the Hartmann solution. Similarly, NaCl solution displayed superior discriminatory capabilities among tissues, with variations of 465%, 157%, and 206%. Distinct spectral modifications were identified, with tissues demonstrating unique responses at each frequency of analysis relative to untreated tissue. Variations in bandwidth alterations were discernible among tissues, providing clear distinctions. In conclusion, the research showed that the crystalloid solution exhibited greater sensitivity and superior tissue contrast at specific frequencies. This study's findings underscore the feasibility of implementing crystalloid solutions to enhance tissue discrimination, similar to the effects of contrast agents.

Wavelength-stepping algorithm for testing the thickness and front and back surfaces of optical plates with high signal-to-noise ratio.

We propose a least-squares phase-stepping algorithm (LS-PSA) consisting of only 14 steps for high-quality optical plate testing. Optical plate testing produces an infinite number of simultaneous fringe patterns due to multiple reflections. However, because of the small reflection of common optical materials, only a few simultaneous fringes have amplitudes above the measuring noise. From these fringes, only the variations of the plate's surfaces and thicknesses are of interest. To measure these plates, one must use wavelength stepping, which corresponds to phase stepping in standard digital interferometry. The designed PSA must phase demodulate a single fringe sequence and filter out the remaining temporal fringes. In the available literature, researchers have adapted PSAs to the dimensions of particular plates. As a consequence, there are as many PSAs published as different testing plate conditions. Moreover, these PSAs are designed with too many phase steps to provide detuning robustness well above the required level. Instead, we mathematically prove that a single 14-step LS-PSA can adapt to several testing setups. As is well known, this 14-step LS-PSA has a maximum signal-to-noise ratio and the highest harmonic rejection among any other 14-step PSA. Due to optical dispersion and experimental length measuring errors, the fringes may have a slight phase detuning. Using propagation error theory, we demonstrate that measuring distances with around 1% uncertainty produces a small and acceptable detuning error for the proposed 14-step LS-PSA.

Affinity-matured homotypic interactions induce spectrum of PfCSP structures that influence protection from malaria infection.

The generation of high-quality antibody responses to Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP), the primary surface antigen of Pf sporozoites, is paramount to the development of an effective malaria vaccine. Here we present an in-depth structural and functional analysis of a panel of potent antibodies encoded by the immunoglobulin heavy chain variable (IGHV) gene IGHV3-33, which is among the most prevalent and potent antibody families induced in the anti-PfCSP immune response and targets the Asn-Ala-Asn-Pro (NANP) repeat region. Cryo-electron microscopy (cryo-EM) reveals a remarkable spectrum of helical antibody-PfCSP structures stabilized by homotypic interactions between tightly packed fragments antigen binding (Fabs), many of which correlate with somatic hypermutation. We demonstrate a key role of these mutated homotypic contacts for high avidity binding to PfCSP and in protection from Pf malaria infection. Together, these data emphasize the importance of anti-homotypic affinity maturation in the frequent selection of IGHV3-33 antibodies and highlight key features underlying the potent protection of this antibody family.

Simultaneous phase and amplitude modulation for dual-sensitivity profilometry of discontinuous objects.

Fringe projection profilometry (FPP) is a well-known technique for digitizing solids. In FPP, straight fringes are projected over a digitizing solid, and a digital camera grabs the projected fringes. The sensitivity of FPP depends on the spatial frequency of the projected fringes. The projected fringes as seen by the camera are phase modulated by the surface of the digitizing object; the demodulated phase is usually wrapped. If the digitizing object has discontinuities larger than the fringe period, the phase jumps are lost. To preserve large phase discontinuities, one must use very low spatial frequency (low-sensitivity) fringes. The drawback of low-sensitivity FPP is that the demodulated phase has low signal-to-noise ratio (SNR). Much higher SNR is obtained by projecting shorter wavelength, at the cost of obtaining wrapped phase. A way out of this problem is to use dual-wavelength FPP (DW-FPP). In DW-FPP, two sets of projected fringes are used, one with long wavelength and another with shorter wavelength. Due to harmonics and gamma distortion, in DW-FPP, one usually needs four phase-shifted fringes for each sensitivity. Here we are proposing to combine the two sensitivities simultaneously, one coded in phase (PM) and the other coded in amplitude (AM), in order to obtain phase and amplitude modulated (DW-PAM) fringes. The low-sensitivity phase is coded as AM of the DW-PAM fringes. The main advantage of DW-PAM fringes is that one reduces the number of phase-shifted fringes by half: instead of using eight phase-shifted fringes (four for low and four for high sensitivities), one would need only four DW-PAM fringes. Of course, if one wants to increase the harmonic rejection of the recovered phase, one may use a higher order phase-shifting algorithm (PSA).

Structural and biophysical correlation of anti-NANP antibodies with in vivo protection against P. falciparum.

The most advanced P. falciparum circumsporozoite protein-based malaria vaccine, RTS,S/AS01 (RTS,S), confers partial protection but with antibody titers that wane relatively rapidly, highlighting the need to elicit more potent and durable antibody responses. Here, we elucidate crystal structures, binding affinities and kinetics, and in vivo protection of eight anti-NANP antibodies derived from an RTS,S phase 2a trial and encoded by three different heavy-chain germline genes. The structures reinforce the importance of homotypic Fab-Fab interactions in protective antibodies and the overwhelmingly dominant preference for a germline-encoded aromatic residue for recognition of the NANP motif. In this study, antibody apparent affinity correlates best with protection in an in vivo mouse model, with the more potent antibodies also recognizing epitopes with repeating secondary structural motifs of type I ß- and Asn pseudo 310 turns; such insights can be incorporated into design of more effective immunogens and antibodies for passive immunization.

Integrative X-ray Structure and Molecular Modeling for the Rationalization of Procaspase-8 Inhibitor Potency and Selectivity.

Caspases are a critical class of proteases involved in regulating programmed cell death and other biological processes. Selective inhibitors of individual caspases, however, are lacking, due in large part to the high structural similarity found in the active sites of these enzymes. We recently discovered a small-molecule inhibitor, 63-R, that covalently binds the zymogen, or inactive precursor (pro-form), of caspase-8, but not other caspases, pointing to an untapped potential of procaspases as targets for chemical probes. Realizing this goal would benefit from a structural understanding of how small molecules bind to and inhibit caspase zymogens. There have, however, been very few reported procaspase structures. Here, we employ X-ray crystallography to elucidate a procaspase-8 crystal structure in complex with 63-R, which reveals large conformational changes in active-site loops that accommodate the intramolecular cleavage events required for protease activation. Combining these structural insights with molecular modeling and mutagenesis-based biochemical assays, we elucidate key interactions required for 63-R inhibition of procaspase-8. Our findings inform the mechanism of caspase activation and its disruption by small molecules and, more generally, have implications for the development of small molecule inhibitors and/or activators that target alternative (e.g., inactive precursor) protein states to ultimately expand the druggable proteome.

Diverse Antibody Responses to Conserved Structural Motifs in Plasmodium falciparum Circumsporozoite Protein.

Malaria vaccine candidate RTS,S/AS01 is based on the central and C-terminal regions of the circumsporozoite protein (CSP) of P. falciparum. mAb397 was isolated from a volunteer in an RTS,S/AS01 clinical trial, and it protects mice from infection by malaria sporozoites. However, mAb397 originates from the less commonly used VH3-15 germline gene compared to the VH3-30/33 antibodies generally elicited by RTS,S to the central NANP repeat region of CSP. The crystal structure of mAb397 with an NPNA4 peptide shows that the central NPNA forms a type I ß-turn and is the main recognition motif. In most anti-NANP antibodies studied to date, a germline-encoded Trp is used to engage the Pro in NPNA ß-turns, but here the Trp interacts with the first Asn. This "conserved" Trp, however, can arise from different germline genes and be located in the heavy or the light chain. Variation in the terminal ψ angles of the NPNA ß-turns results in different dispositions of the subsequent NPNA and, hence, different stoichiometries and modes of antibody binding to rsCSP. Diverse protective antibodies against NANP repeats are therefore not limited to a single germline gene response or mode of binding.

Fourier spectra for nonuniform phase-shifting algorithms based on principal component analysis.

We develop an error-free nonuniform phase-stepping algorithm (nPSA) based on principal component analysis (PCA). PCA-based algorithms typically give phase-demodulation errors when applied to nonuniform phase-shifted interferograms. We present a straightforward way to correct those PCA phase-demodulation errors. We give mathematical formulas to fully analyze PCA-based nPSA (PCA-nPSA). These formulas give a) the PCA-nPSA frequency transfer function (FTF), b) its corrected Lissajous figure, c) the corrected PCA-nPSA formula, d) its harmonic robustness (RH), and e) its signal-to-noise-ratio (SNR). We show that the PCA-nPSA can be seen as a linear quadrature filter and, as consequence, one can find its FTF. Using the FTF, we show why plain PCA often fails to demodulate nonuniform phase-shifted fringes. Previous works on PCA-nPSA (without FTF), give specific numerical/experimental fringe data to "visually demonstrate" that their new nPSA works better than its competitors. This often leads to biased/favorable fringe pattern selections which "visually demonstrate" the superior performance of their new nPSA. This biasing is herein totally avoided because we provide figures-of-merit formulas based on linear systems and stochastic process theories. However, and for illustrative purposes only, we provide specific fringe data phase-demodulation, including comprehensive analysis and comparisons.

Selective and Rapid Cell-Permeable Inhibitor of Human Caspase-3.

Individual roles and overlapping functionalities of 12 human caspases during apoptosis and other cellular processes remain poorly resolved primarily due to a lack of chemical tools. Here we present a new selective caspase-3 inhibitor, termed Ac-ATS010-KE, with rapid and irreversible binding kinetics. Relative to previously designed caspase-3-selective molecules that have tremendously abated inhibitory rates and thus limited use in biological settings, the improved kinetics of Ac-ATS010-KE permits its use in a cell-based capacity. We demonstrate that Ac-ATS010-KE prevents apoptosis with comparable efficacy to the general caspase inhibitor Ac-DEVD-KE and surprisingly does so without side-chain methylation. This observation is in contrast to the well-established peptide modification strategy typically employed for improving cellular permeability. Ac-ATS010-KE protects against extrinsic apoptosis, which demonstrates the utility of a thiophene carboxylate leaving group in biological settings, challenges the requisite neutralization of free carboxylic acids to improve cell permeability, and provides a tool-like compound to interrogate the role of caspase-3 in a variety of cellular processes.

X-ray structure of an inactive zymogen clostripain-like protease from Parabacteroides distasonis.

The clostripain-like (C11) family of cysteine proteases are ubiquitously produced by the vast majority of the bacterial strains that make up the human distal gut microbiome. Recent reports show that some C11 proteases promote host immune responses and bacterial pathogenesis, including the induction of neutrophil phagocytosis and the activation of bacterial pathogenic toxins, respectively. The crystal structure of distapain, the only C11 protease predicted within the genome of the commensal bacterium Parabacteroides distasonis, was determined in the inactive zymogen state to 1.65 Šresolution. This is the first C11 protease structure of a zymogen, and the structure helped to uncover key unique conformations among critical active-site residues that are likely to assist in preserving the inactive protease. His135, a member of the catalytic dyad, is repositioned approximately 5.5 Šfrom the orientation found in active C11 structures and forms a hydrogen bond to Asp180 and a π-stacking interaction with Trp133. The structure sheds light on the potential importance of Asp180 and Trp133, as these residues are highly conserved across C11 proteases. Structure elucidation of C11 proteases will ultimately help to identify new ways to chemically and/or biologically regulate this family of enzymes, which represent potential drug-discovery targets in microbiome-related gastrointestinal diseases.

Design of nonlinearly spaced phase-shifting algorithms using their frequency transfer function.

Here we show how to design phase-shifting algorithms (PSAs) for nonuniform/nonlinear (NL) phase-shifted fringe patterns using their frequency transfer function (FTF). Assuming that the NL phase steps are known, we introduce the desired zeroes in the FTF to obtain the specific NL-PSA formula. The advantage of designing NL-PSAs based on their FTF is that one can reject many distorting harmonics of the fringes. We can also estimate the signal-to-noise ratio for interferograms corrupted by additive white Gaussian noise. Finally, for non-distorted noiseless fringes, the proposed NL-PSA retrieves the modulating phase error free, just as standard/linear PSAs do.

X-ray Structures of Two Bacteroides thetaiotaomicron C11 Proteases in Complex with Peptide-Based Inhibitors.

Commensal bacteria secrete proteins and metabolites to influence host intestinal homeostasis, and proteases represent a significant constituent of the components at the host:microbiome interface. Here, we determined the structures of the two secreted C11 cysteine proteases encoded by the established gut commensal Bacteroides thetaiotaomicron. We employed mutational analysis to demonstrate the two proteases, termed "thetapain" and "iotapain", undergo in trans autoactivation after lysine and/or arginine residues, as observed for other C11 proteases. We determined the structures of the active forms of thetapain and iotapain in complex with irreversible peptide inhibitors, Ac-VLTK-AOMK and biotin-VLTK-AOMK, respectively. Structural comparisons revealed key active-site interactions important for peptide recognition are more extensive for thetapain; however, both proteases employ a glutamate residue to preferentially bind small polar residues at the P2 position. Our results will aid in the design of protease-specific probes to ultimately understand the biological role of C11 proteases in bacterial fitness, elucidate their host and/or microbial substrates, and interrogate their involvement in microbiome-related diseases.

Substrate Profiling and High Resolution Co-complex Crystal Structure of a Secreted C11 Protease Conserved across Commensal Bacteria.

Cysteine proteases are among the most abundant hydrolytic enzymes produced by bacteria, and this diverse family of proteins have significant biological roles in bacterial viability and environmental interactions. Members of the clostripain-like (C11) family of cysteine proteases from commensal gut bacterial strains have recently been shown to mediate immune responses by inducing neutrophil phagocytosis and activating bacterial pathogenic toxins. Development of substrates, inhibitors, and probes that target C11 proteases from enteric bacteria will help to establish the role of these proteins at the interface of the host and microbiome in health and disease. We employed a mass spectrometry-based substrate profiling method to identify an optimal peptide substrate of PmC11, a C11 protease secreted by the commensal bacterium Parabacteroides merdae. Using this substrate sequence information, we synthesized a panel of fluorogenic substrates to calculate kcat and KM and to evaluate the importance of the P2 amino acid for substrate turnover. A potent and irreversible tetrapeptide inhibitor with a C-terminal acyloxymethyl ketone warhead, Ac-VLTK-AOMK, was then synthesized. We determined the crystal structure of PmC11 in complex with this inhibitor and uncovered key active-site interactions that govern PmC11 substrate recognition and specificity. This is the first C11 protease structure in complex with a substrate mimetic and is also the highest resolution crystal structure of a C11 protease to date at 1.12 Å resolution. Importantly, subjecting human epithelial cell lysates to PmC11 hydrolysis in combination with subtiligase-based N-terminal labeling and tandem mass spectrometry proteomics complemented the stringent substrate specificity observed in the in vitro substrate profiling experiment. The combination of chemical biological, biophysical, and biochemical techniques presented here to elucidate and characterize PmC11 substrate selectivity can be expanded to other proteases and the development of chemical tools to study these essential proteins in biologically relevant samples, such as the highly complex distal gut microbiome.

Proteome-wide covalent ligand discovery in native biological systems.

Small molecules are powerful tools for investigating protein function and can serve as leads for new therapeutics. Most human proteins, however, lack small-molecule ligands, and entire protein classes are considered 'undruggable'. Fragment-based ligand discovery can identify small-molecule probes for proteins that have proven difficult to target using high-throughput screening of complex compound libraries. Although reversibly binding ligands are commonly pursued, covalent fragments provide an alternative route to small-molecule probes, including those that can access regions of proteins that are difficult to target through binding affinity alone. Here we report a quantitative analysis of cysteine-reactive small-molecule fragments screened against thousands of proteins in human proteomes and cells. Covalent ligands were identified for >700 cysteines found in both druggable proteins and proteins deficient in chemical probes, including transcription factors, adaptor/scaffolding proteins, and uncharacterized proteins. Among the atypical ligand-protein interactions discovered were compounds that react preferentially with pro- (inactive) caspases. We used these ligands to distinguish extrinsic apoptosis pathways in human cell lines versus primary human T cells, showing that the former is largely mediated by caspase-8 while the latter depends on both caspase-8 and -10. Fragment-based covalent ligand discovery provides a greatly expanded portrait of the ligandable proteome and furnishes compounds that can illuminate protein functions in native biological systems.

High-resolution bispectral imager at 1000 frames per second.

We describe a bispectral, 1000-frames per second imaging instrument working simultaneously in two spectral bands. These bands may be selected for a specific application; however, we implement a pair centered at 4.3 µm and 4.66 µm. Synchronization is accomplished by employing a single focal plane array. To demonstrate the performance of the bispectral imager, we apply it to the methane flame of a Bunsen burner in a near conjugate configuration with flame image length subtending at about 200 pixels. The instrument detects bispectral puffing at 2 Hz, pulsations, and bispectral radiation oscillations, first reported here in two spectral intervals. The period of oscillatory spectral components in two bands is the same, about 3 Hz for this flame, with delay of a quarter period between them, first reported here. With 1-ms integration time, we detect significant formation of turbulence and vortices, especially pronounced in the region where the flame transitions into a plume. We display bispectral ratioed images of flames in near-real time with either the laboratory or the field device.

Identification and Co-complex Structure of a New S. pyogenes SpeB Small Molecule Inhibitor.

The secreted Streptococcus pyogenes cysteine protease SpeB is implicated in host immune system evasion and bacterial virulence. We present a small molecule inhibitor of SpeB 2477 identified from a high-throughput screen based on the hydrolysis of a fluorogenic peptide substrate Ac-AIK-AMC. 2477 inhibits other SpeB-related proteases but not human caspase-3, suggesting that the molecule targets proteases with the papain-like structural fold. A 1.59 Å X-ray crystal structure of 2477 bound to the SpeB active site reveals the mechanism of inhibition and the essential constituents of 2477 necessary for binding. An assessment against a panel of 2477 derivatives confirms our structural findings and shows that a carbamate and nitrile on 2477 are required for SpeB inhibition, as these moieties provide an extensive network of electrostatic and hydrogen-bonding interactions with SpeB active site residues. Surprisingly, despite 2477 having a reduced inhibitory potential against papain, the majority of 2477-related compounds inhibit papain to a much greater and broader extent than SpeB. These findings indicate that SpeB is more stringently selective than papain for this panel of small molecule inhibitors. On the basis of our structural and biochemical characterization, we propose modifications to 2477 for subsequent rounds of inhibitor design that will impart specificity to SpeB over other papain-like proteases, including alterations of the compound to exploit the differences in CA protease active site pocket sizes and electrostatics.

Wrapping-free phase retrieval with applications to interferometry, 3D-shape profiling, and deflectometry.

Phase unwrapping is probably the most challenging step in the phase retrieval process in phase-shifting and spatial-carrier interferometry. Likewise, phase unwrapping is required in 3D-shape profiling and deflectometry. In this paper, we present a novel phase retrieval method that completely sidesteps the phase unwrapping process, significantly eliminating the guessing in phase reconstruction and thus decreasing the time data processing. The proposed wrapping-free method is based on the direct integration of the spatial derivatives of the interference patterns under the single assumption that the phase is continuous. This assumption is valid in most physical applications. Validation experiments are presented confirming the robustness of the proposed method.

Proteinases in excretory-secretory products of Toxocara canis second-stage larvae: zymography and modeling insights.

Components released in excretory-secretory products of Toxocara canis larvae (TES) include phosphatidylethanolamine-binding proteins (TES26), mucins (TES120, MUC2-5), and C-type lectins (TES32, TES70) and their biochemical, immunological, and diagnostic properties have been extensively studied albeit proteinase activities towards physiological substrates are almost unknown. Proteolytic activities in TES samples were first analyzed by gel electrophoresis with gelatin as substrate. Major activities of ~400, 120, and 32 kDa in TES were relatively similar over a broad pH range (5.5-9.0) and all these were of the serine-type as leupeptin abolished gelatinolysis. Further, the ~400 kDa component degraded all physiological substrates tested (laminin, fibronectin, albumin, and goat IgG) and the 120 kDa component degraded albumin and goat IgG while proteinases of lower MW (45, 32, and 26 kDa) only degraded laminin and fibronectin, preferentially at alkaline pH (9.0). By protein modeling approaches using the known sequences of TES components, only TES26 and MUC4 displayed folding patterns significantly related to reference serine proteinases. These data suggest that most of serine proteinase activities secreted in vitro by infective larvae of T. canis have intriguing nature but otherwise help the parasite to affect multiple components of somatic organs and bodily fluids within the infected host.

Selective inhibition of initiator versus executioner caspases using small peptides containing unnatural amino acids.

Caspases are fundamental to many essential biological processes, including apoptosis, differentiation, and inflammation. Unregulated caspase activity is also implicated in the development and progression of several diseases, such as cancer, neurodegenerative disorders, and sepsis. Unfortunately, it is difficult to determine exactly which caspase(s) of the 11 isoforms that humans express is responsible for specific biological functions. This lack of resolution is primarily due to highly homologous active sites and overlapping substrates. Currently available peptide-based inhibitors and probes are based on specificity garnered from peptide substrate libraries. For example, the canonical tetrapeptide LETD was discovered as the canonical sequence that is optimally recognized by caspase-8; however, LETD-based inhibitors and substrates promiscuously bind to other isoforms with equal affinity, including caspases-3, -6, and -9. In order to mitigate this problem, we report the identification of a new series of compounds that are >100-fold selective for inhibiting the initiator caspases-8 and -9 over the executioner caspases-3, -6, and -7.

Discovery of a highly selective caspase-3 substrate for imaging live cells.

Caspases are a family of cysteine proteases that are well-known for their roles in apoptosis and inflammation. Recent studies provide evidence that caspases are also integral to many additional cellular processes, such as differentiation and proliferation. Likewise, aberrant caspase activity has been implicated in the progression of several diseases, including neurodegenerative disorders, cancer, cardiovascular disease, and sepsis. These observations establish the importance of caspases to a diverse array of physiological functions and future endeavors will undoubtedly continue to elucidate additional processes that require caspase activity. Unfortunately, the existence of 11 functional human caspases, with overlapping substrate specificities, confounds the ability to confidently assign one or more isoforms to biological phenomena. Herein, we characterize a first-in-class FRET substrate that is selectively recognized by active caspase-3 over other initiator and executioner caspases. We further apply this substrate to specifically image caspase-3 activity in live cells undergoing apoptosis.