Nature-Inspired Gallinamides Are Potent Antischistosomal Agents: Inhibition of the Cathepsin B1 Protease Target and Binding Mode Analysis.

Schistosomiasis, caused by a parasitic blood fluke of the genus Schistosoma, is a global health problem for which new chemotherapeutic options are needed. We explored the scaffold of gallinamide A, a natural peptidic metabolite of marine cyanobacteria that has previously been shown to inhibit cathepsin L-type proteases. We screened a library of 19 synthetic gallinamide A analogs and identified nanomolar inhibitors of the cathepsin B-type protease SmCB1, which is a drug target for the treatment of schistosomiasis mansoni. Against cultured S. mansoni schistosomula and adult worms, many of the gallinamides generated a range of deleterious phenotypic responses. Imaging with a fluorescent-activity-based probe derived from gallinamide A demonstrated that SmCB1 is the primary target for gallinamides in the parasite. Furthermore, we solved the high-resolution crystal structures of SmCB1 in complex with gallinamide A and its two analogs and describe the acrylamide covalent warhead and binding mode in the active site. Quantum chemical calculations evaluated the contribution of individual positions in the peptidomimetic scaffold to the inhibition of the target and demonstrated the importance of the P1' and P2 positions. Our study introduces gallinamides as a powerful chemotype that can be exploited for the development of novel antischistosomal chemotherapeutics.

Development and Application of Control Concepts for Twin-Screw Wet Granulation in the ConsiGmaTM-25: Part 2 Granule Size.

Traditional operation modes, such as running the production processes at constant process settings or within a narrow design space, do not fully exploit the advantages of continuous pharmaceutical manufacturing. Integrating Quality by Control (QbC) algorithms as a standard component of production processes can mitigate the effect of diverse process disturbances and enhance process efficiency, particularly in terms of production costs and environmental footprint. This paper explores the potential of QbC algorithms for optimizing twin-screw wet granulation in the ConsiGmaTM-25 manufacturing line, specifically addressing granule size. It represents the second part of a study (Celikovic et al. (2024)) focused on granule composition. The concepts proposed in this work rely on process analytical technology (PAT) equipment for real-time monitoring of the granulation CQAs and a dynamic process model linking the granulation process parameters and the monitored CQAs. The granule size model identified via the local-linear-model-tree (LoLiMoT) algorithm is used to develop both a model predictive controller (MPC) and a granule size soft sensor. The MPC employs this model as a core component for selecting optimal granulation parameters to ensure the production of granules with target size. A digital operator assistant is developed to address disturbances that cannot be mitigated via MPC but can be eliminated by the plant operators. This study systematically outlines a workflow, starting from conceptualization, moving through simulation development, and finally ending with real-world application on a production line. In this final step, all proposed concepts are transferred to the ConsiGmaTM-25 manufacturing line, where their performance is validated through selected disturbance scenarios.

Development and Application of Control Concepts for Twin-Screw Wet Granulation in the ConsiGmaTM-25: Part 1 Granule Composition.

Continuous manufacturing of pharmaceuticals offers several benefits, such as increased production efficiency, enhanced product quality control, and lower environmental footprint. To fully exploit these benefits, standard operation mode (production processes with no or minimal disturbance mitigation measures) should be supported by adopting novel quality-by-control (QbC) methodologies. The paper at hand is the first part of a study focused on developing QbC algorithms for optimizing twin-screw wet granulation in the industrial manufacturing line ConsiGmaTM-25, specifically addressing granule composition. This work relies on previously established process-analytical-technology (PAT) equipment for real-time monitoring of the granule composition, i.e., the active pharmaceutical ingredient (API) and liquid content in wet granules. The developed control platform integrates model-based process control algorithms that aim to keep the API- and liquid content at target values through real-time adjustments of the process parameters. Furthermore, the platform integrates a digital operator assistant, which aims to detect and classify granulation disturbances and provides messages and instructions for the plant operator. The present manuscript systematically outlines all design steps from the development phase in the simulation environment to the final real system application and validation. The control platform's performance is demonstrated through selected test scenarios on the ConsiGmaTM-25 manufacturing line. The obtained results indicate improved disturbance robustness and an increase in intermediate/final product quality (compared to conventional operating modes): The process control algorithms successfully maintained the API- and liquid content at target values despite process disturbances. Furthermore, realistic disturbances (feeder, pump, and material) were accurately detected and classified by the digital assistant algorithm. The information was provided through a user interface, offering real-time support for plant personnel.

An Unusual Two-Domain Thyropin from Tick Saliva: NMR Solution Structure and Highly Selective Inhibition of Cysteine Cathepsins Modulated by Glycosaminoglycans.

The structure and biochemical properties of protease inhibitors from the thyropin family are poorly understood in parasites and pathogens. Here, we introduce a novel family member, Ir-thyropin (IrThy), which is secreted in the saliva of Ixodes ricinus ticks, vectors of Lyme borreliosis and tick-borne encephalitis. The IrThy molecule consists of two consecutive thyroglobulin type-1 (Tg1) domains with an unusual disulfide pattern. Recombinant IrThy was found to inhibit human host-derived cathepsin proteases with a high specificity for cathepsins V, K, and L among a wide range of screened cathepsins exhibiting diverse endo- and exopeptidase activities. Both Tg1 domains displayed inhibitory activities, but with distinct specificity profiles. We determined the spatial structure of one of the Tg1 domains by solution NMR spectroscopy and described its reactive center to elucidate the unique inhibitory specificity. Furthermore, we found that the inhibitory potency of IrThy was modulated in a complex manner by various glycosaminoglycans from host tissues. IrThy was additionally regulated by pH and proteolytic degradation. This study provides a comprehensive structure-function characterization of IrThy-the first investigated thyropin of parasite origin-and suggests its potential role in host-parasite interactions at the tick bite site.

Beyond Empathy to System Change: Four Poems on Health by Bertolt Brecht.

Bertolt Brecht's poem "A Worker's Speech to a Doctor" is frequently cited as a means to raise awareness among health workers of the health effects of living and working conditions. Less cited is his Call to Arms trilogy of poems, which calls for class-based action to transform the capitalist economic system that sickens and kills so many. In this article, we show how "A Worker's Speech to a Doctor," with its plea for empathy for the ill, contrasts with the more activist and often militant tone of the Call to Arms trilogy: "Call to a Sick Communist," "The Sick Communist's Answer to the Comrades," and "Call to the Doctors and Nurses." We also show that, while "A Worker's Speech to a Doctor" has been applied in the training of health workers, its accusatorial tone towards health workers' complicity in the system the poem is critiquing risks alienating such workers. In contrast, the Call to Arms trilogy seeks common ground, inviting these same workers into the broader political and social fight against injustice. While we contend that the description of the sick worker as a "Communist" risks estranging these health workers, our analysis of the Call to Arms poems nevertheless indicates that their use can contribute to moving health workers' educational discourse beyond a laudable but fleeting elicitation of empathy for the ill towards a structural critique and deeper systemic understanding in order to prompt action by health workers to reform or even replace the capitalist economic system that sickens and kills so many.

Comparison of subthalamic unilateral and bilateral theta burst deep brain stimulation in Parkinson's disease.

High-frequency, conventional deep brain stimulation (DBS) of the subthalamic nucleus (STN) in Parkinson's disease (PD) is usually applied bilaterally under the assumption of additive effects due to interhemispheric crosstalk. Theta burst stimulation (TBS-DBS) represents a new patterned stimulation mode with 5 Hz interburst and 200 Hz intraburst frequency, whose stimulation effects in a bilateral mode compared to unilateral are unknown. This single-center study evaluated acute motor effects of the most affected, contralateral body side in 17 PD patients with unilateral subthalamic TBS-DBS and 11 PD patients with bilateral TBS-DBS. Compared to therapy absence, both unilateral and bilateral TBS-DBS significantly improved (p < 0.05) lateralized Movement Disorder Society-Unified Parkinson's Disease Rating Scale part III (MDS-UPDRS III) scores. Bilateral TBS-DBS revealed only slight, but not significant additional effects in comparison to unilateral TBS-DBS on total lateralized motor scores, but on the subitem lower limb rigidity. These results indicate that bilateral TBS-DBS has limited additive beneficial effects compared to unilateral TBS-DBS in the short term.

Insight Into the Dynamics of the Ixodes ricinus Nymphal Midgut Proteome.

Ticks are ectoparasites that feed on blood and have an impressive ability to consume and process enormous amounts of host blood, allowing extremely long periods of starvation between blood meals. The central role in the parasitic lifestyle of ticks is played by the midgut. This organ efficiently stores and digests ingested blood and serves as the primary interface for the transmission of tick-borne pathogens. In this study, we used a label-free quantitative approach to perform a novel dynamic proteomic analysis of the midgut of Ixodesricinus nymphs, covering their development from unfed to pre-molt stages. We identified 1534 I. ricinus-specific proteins with a relatively low proportion of host proteins. This proteome dataset, which was carefully examined by manual scrutiny, allowed precise annotation of proteins important for blood meal processing and their dynamic changes during nymphal ontogeny. We focused on midgut molecules related to lipid hydrolysis, storage, and transport, opening a yet unexplored avenue for studying lipid metabolism in ticks. Further dynamic profiling of the tick's multi-enzyme digestive network, protease inhibitors, enzymes involved in redox homeostasis and detoxification, antimicrobial peptides, and proteins responsible for midgut colonization by Borrelia spirochetes promises to uncover new targets for targeting tick nymphs, the most critical life stage for transmission the pathogens that cause tick-borne diseases.

A system theory based digital model for predicting the cumulative fluid balance course in intensive care patients.

Background: Surgical interventions can cause severe fluid imbalances in patients undergoing cardiac surgery, affecting length of hospital stay and survival. Therefore, appropriate management of daily fluid goals is a key element of postoperative intensive care in these patients. Because fluid balance is influenced by a complex interplay of patient-, surgery- and intensive care unit (ICU)-specific factors, fluid prediction is difficult and often inaccurate. Methods: A novel system theory based digital model for cumulative fluid balance (CFB) prediction is presented using recorded patient fluid data as the sole parameter source by applying the concept of a transfer function. Using a retrospective dataset of n = 618 cardiac intensive care patients, patient-individual models were created and evaluated. RMSE analyses and error calculations were performed for reasonable combinations of model estimation periods and clinically relevant prediction horizons for CFB. Results: Our models have shown that a clinically relevant time horizon for CFB prediction with the combination of 48 h estimation time and 8-16 h prediction time achieves high accuracy. With an 8-h prediction time, nearly 50% of CFB predictions are within ±0.5 L, and 77% are still within the clinically acceptable range of ±1.0 L. Conclusion: Our study has provided a promising proof of principle and may form the basis for further efforts in the development of computational models for fluid prediction that do not require large datasets for training and validation, as is the case with machine learning or AI-based models. The adaptive transfer function approach allows estimation of CFB course on a dynamically changing patient fluid balance system by simulating the response to the current fluid management regime, providing a useful digital tool for clinicians in daily intensive care.

Transition moments beyond the electric-dipole approximation: Visualization and basis set requirements.

In the simulation of x-ray absorption spectroscopy, the validity of the electric-dipole approximation comes into question. Three different schemes exist to go beyond this approximation: the first scheme is based on the full semi-classical light-matter interaction, whereas the latter two schemes, referred to as the generalized length and velocity representation, are based on truncated multipole expansions. Even though these schemes have been successfully implemented in several quantum chemistry codes, their basis set requirements remained largely unknown. Here, we assess basis set requirements of these three schemes. We have considered 1s1/2 and 7s1/2 → 7p1/2 transitions in the radium atom, representative of core and valence excitations, respectively, and carried out calculations with dyall.aeXz (X = 2, 3, 4) basis sets at the four-component relativistic TD-HF level of theory. Our basis set study was greatly facilitated by the generation and visualization of radial distributions of transition moment densities, allowing for a straightforward comparison with equivalent finite-difference calculations. Pertaining to the truncated interaction, we find that the length representation electric multipole is the easiest to converge, requiring the dyall.ae2z basis for low-order multipoles and the dyall.ae4z basis at higher orders. The magnetic multipole moments follow a similar trend although they are more difficult to converge. The velocity representation electric multipoles are the most difficult to converge: at high orders, the dyall.ae3z and dyall.ae4z basis sets introduce artificial peaks and oscillations, which increase the overall error. These artifacts are associated with linear dependence issues in the small component space of larger basis sets. The full interaction operator, however, does not suffer from these problems, and we therefore recommend its use in the simulation of x-ray spectroscopy.

Control oriented modeling of twin-screw granulation in the ConsiGmaTM-25 production plant.

ConsiGmaTM-25 is a continuous production plant integrating a twin-screw granulation, fluid bed drying, granule conditioning, and a tableting unit. The particle size distribution (PSD), active pharmaceutical ingredient (API) content, and liquid content of wet granules after twin-screw granulation affect the quality of intermediate and final products. This paper proposes methods for real-time monitoring of these quantities and control-oriented modeling of the granulator. The PSD of wet granules is monitored via an in-line process analytical technology (PAT) probe based on the spatial velocimetry principle. The algorithm for signal processing and evaluation of PSD characteristics is developed and applied to the acquired PSD data. A dynamic process model predicting PSD characteristics from granulation parameters is trained via the local linear model tree (LoLiMoT) approach. The experimental data required for the model training are collected via systematically designed excitation runs. Finally, the performance of the identified model is examined and verified by means of a new set of validation runs. Furthermore, an in-line PAT probe based on Raman spectroscopy is developed and integrated after the granulator. The API- and liquid content of produced wet granules are evaluated from the spectral data by means of chemometric modeling, and chemometric models are validated on a separate set of experimental data. The solutions proposed in this research can be used as a reliable (and necessary) basis for the development of advanced quality-by-design control concepts (e.g., PSD process control). Such concepts would ultimately improve the ConsiGmaTM-25 process performance in terms of robustness against disturbances and quality of intermediate and final products.

An evolutionary molecular adaptation of an unusual stefin from the liver fluke Fasciola hepatica redefines the cystatin superfamily.

Fasciolosis is a worldwide parasitic disease of ruminants and an emerging human disease caused by the liver fluke Fasciola hepatica. The cystatin superfamily of cysteine protease inhibitors is composed of distinct families of intracellular stefins and secreted true cystatins. FhCyLS-2 from F. hepatica is an unusual member of the superfamily, where our sequence and 3D structure analyses in this study revealed that it combines characteristics of both families. The protein architecture demonstrates its relationship to stefins, but FhCyLS-2 also contains the secretion signal peptide and disulfide bridges typical of true cystatins. The secretion status was confirmed by detecting the presence of FhCyLS-2 in excretory/secretory products, supported by immunolocalization. Our high-resolution crystal structure of FhCyLS-2 showed a distinct disulfide bridging pattern and functional reactive center. We determined that FhCyLS-2 is a broad specificity inhibitor of cysteine cathepsins from both the host and F. hepatica, suggesting a dual role in the regulation of exogenous and endogenous proteolysis. Based on phylogenetic analysis that identified several FhCyLS-2 homologues in liver/intestinal foodborne flukes, we propose a new group within the cystatin superfamily called cystatin-like stefins.

Proteases and their inhibitors involved in Schistosoma mansoni egg-host interaction revealed by comparative transcriptomics with Fasciola hepatica eggs.

Schistosoma mansoni eggs are the main causative agents of the pathological manifestations of schistosomiasis. The eggs are laid in the host bloodstream, then they migrate through the intestinal wall into the lumen. However, a significant proportion of the eggs become lodged in the liver, where they cause inflammation and fibrosis. In this study, we focus on a specific group of proteins expressed by the egg, namely proteases and their inhibitors. These molecules are often involved in schistosome-host interactions, but are still unexplored in the egg stage. Using RNA-seq and comparative transcriptomics of immature and mature S. mansoni eggs, we mapped the portfolio of proteases and their inhibitors, and determined their gene expression levels. In addition, we compared these data with gene expression of proteases and their inhibitors in Fasciola hepatica eggs. Fasciola hepatica eggs served as a useful comparative model, as they do not migrate through tissues and inflict pathology. We detected transcription of 135 and 117 proteases in S. mansoni and F. hepatica eggs, respectively, with 87 identified as orthologous between the two species. In contrast, we observed only four orthologous inhibitors out of 21 and 16 identified in S. mansoni and F. hepatica eggs, respectively. Among others, we measured high and developmentally regulated levels of expression of metalloproteases in S. mansoni eggs, specifically aminopeptidase N1, endothelin-converting enzyme 1, and several leishmanolysin-like peptidases. We identified highly transcribed protease inhibitors serpin and alpha-2-macroglobulin that are unique to S. mansoni eggs, and antistasin-like inhibitor in F. hepatica eggs. This study provides new insights into the portfolio of proteases and inhibitors expressed by S. mansoni with potential roles in egg tissue migration, stimulation of angiogenesis, and interaction with host blood and immunity.

Subthalamic and nigral neurons are differentially modulated during parkinsonian gait.

The parkinsonian gait disorder and freezing of gait are therapeutically demanding symptoms with considerable impact on quality of life. The aim of this study was to assess the role of subthalamic and nigral neurons in the parkinsonian gait control using intraoperative microelectrode recordings of basal ganglia neurons during a supine stepping task. Twelve male patients (56 ± 7 years) suffering from moderate idiopathic Parkinson's disease (disease duration 10 ± 3 years, Hoehn and Yahr stage 2), undergoing awake neurosurgery for deep brain stimulation, participated in the study. After 10 s resting, stepping at self-paced speed for 35 s was followed by short intervals of stepping in response to random 'start' and 'stop' cues. Single- and multi-unit activity was analysed offline in relation to different aspects of the stepping task (attentional 'start' and 'stop' cues, heel strikes, stepping irregularities) in terms of firing frequency, firing pattern and oscillatory activity. Subthalamic nucleus and substantia nigra neurons responded to different aspects of the stepping task. Of the subthalamic nucleus neurons, 24% exhibited movement-related activity modulation as an increase of the firing rate, suggesting a predominant role of the subthalamic nucleus in motor aspects of the task, while 8% of subthalamic nucleus neurons showed a modulation in response to the attentional cues. In contrast, responsive substantia nigra neurons showed activity changes exclusively associated with attentional aspects of the stepping task (15%). The firing pattern of subthalamic nucleus neurons revealed gait-related firing regularization and a drop of beta oscillations during the stepping performance. During freezing episodes instead, there was a rise of beta oscillatory activity. This study shows for the first time specific, task-related subthalamic nucleus and substantia nigra single-unit activity changes during gait-like movements in humans with differential roles in motor and attentional control of gait. The emergence of perturbed firing patterns in the subthalamic nucleus indicates a disrupted information transfer within the gait network, resulting in freezing of gait.

Two Tags in One Probe: Combining Fluorescence- and Biotin-based Detection of the Trypanosomal Cysteine Protease Rhodesain.

Rhodesain is the major cysteine protease of the protozoan parasite Trypanosoma brucei and a therapeutic target for sleeping sickness, a fatal neglected tropical disease. We designed, synthesized and characterized a bimodal activity-based probe that binds to and inactivates rhodesain. This probe exhibited an irreversible mode of action and extraordinary potency for the target protease with a kinac /Ki value of 37,000 M-1 s-1 . Two reporter tags, a fluorescent coumarin moiety and a biotin affinity label, were incorporated into the probe and enabled highly sensitive detection of rhodesain in a complex proteome by in-gel fluorescence and on-blot chemiluminescence. Furthermore, the probe was employed for microseparation and quantification of rhodesain and for inhibitor screening using a competition assay. The developed bimodal rhodesain probe represents a new proteomic tool for studying Trypanosoma pathobiochemistry and antitrypanosomal drug discovery.

Transcriptomic and proteomic profiling of peptidase expression in Fasciola hepatica eggs developing at host's body temperature.

Fasciola hepatica is a global parasite of livestock which also causes a neglected zoonosis in humans. The parasite's communication with the host during its complicated lifecycle is based on an ingenious enzymatic apparatus which includes a variety of peptidases. These enzymes are implicated in parasite migration, pathogenesis of the disease, and modification of host immune response. Although the dynamics of proteolytic machinery produced by intra-mammalian F. hepatica life stages has been previously investigated in great detail, peptidases of the eggs so far received little scientific attention. In this study, we performed a comparative RNA-seq analysis aimed at identification of peptidases expressed in F. hepatica eggs, cultured at 37 °C to represent gall bladder retained eggs, for different time periods and employed mass spectrometry in order to identify and quantify peptidases translated in F. hepatica egg lysates. We demonstrated that F. hepatica eggs undergo significant molecular changes when cultured at the physiological temperature of the definitive host. Egg transcriptome is subject to numerous subtle changes while their proteome is even more variable. The peptidase profile is considerably modified on both transcriptome and proteome level. Finally, we measured and classified proteolytic activities in extracts from F. hepatica eggs using a library of fluorogenic substrates and peptidase class-selective inhibitors. Activities of threonine peptidases were detected constantly, while the cysteine peptidases prevailing in freshly laid eggs are substituted by aspartic peptidase and metallopeptidase activities in the later stages of egg development.

Highly potent inhibitors of cathepsin K with a differently positioned cyanohydrazide warhead: structural analysis of binding mode to mature and zymogen-like enzymes.

Cathepsin K (CatK) is a target for the treatment of osteoporosis, arthritis, and bone metastasis. Peptidomimetics with a cyanohydrazide warhead represent a new class of highly potent CatK inhibitors; however, their binding mechanism is unknown. We investigated two model cyanohydrazide inhibitors with differently positioned warheads: an azadipeptide nitrile Gü1303 and a 3-cyano-3-aza-ß-amino acid Gü2602. Crystal structures of their covalent complexes were determined with mature CatK as well as a zymogen-like activation intermediate of CatK. Binding mode analysis, together with quantum chemical calculations, revealed that the extraordinary picomolar potency of Gü2602 is entropically favoured by its conformational flexibility at the nonprimed-primed subsites boundary. Furthermore, we demonstrated by live cell imaging that cyanohydrazides effectively target mature CatK in osteosarcoma cells. Cyanohydrazides also suppressed the maturation of CatK by inhibiting the autoactivation of the CatK zymogen. Our results provide structural insights for the rational design of cyanohydrazide inhibitors of CatK as potential drugs.

Probing chirality across the electromagnetic spectrum with the full semi-classical light-matter interaction.

We present a formulation and implementation of anisotropic and isotropic electronic circular dichroism (ECD) using the full semi-classical light-matter interaction operator within a four-component relativistic framework. Our treatment uniquely accounts for both beyond-first-order light-matter interactions and relativistic effects, enabling us to investigate the ECD response across the electromagnetic spectrum from optical to x-ray wavelengths where relativistic selection rules and spatial field variations gain increasing importance. We consider the isotropic and oriented ECD across the valence transition and sulfur L- and K-edge transitions in the simplest disulfides, H2S2 and (CH3S)2, and evaluate the influence of the full interaction by comparing to a traditional truncated formulation in the Coulomb gauge (velocity representation). Additionally, we demonstrate that in the relativistic formalism, it is possible to work in the velocity representation, hence keeping order-by-order gauge-origin invariance, contrary to the multipolar gauge, yet being able to distinguish electric and magnetic multipole contributions. Going beyond a first-order treatment in the wave vector is mandatory in the higher-energy end of the soft x-ray region and beyond where the consequent intensity redistribution becomes significant. While the sulfur K-edge absorption spectrum is essentially unaffected by this redistribution, the signed differential counterpart is not: At least third-order contributions are required to describe the differential absorption profile that is otherwise overestimated by a factor of about two. The first-order description deteriorates at higher transition energies (beyond ∼1000 eV) where it may even fail to predict the sign of individual differential oscillator strengths.

An Activity-Based Probe for Cathepsin K Imaging with Excellent Potency and Selectivity.

The cysteine protease cathepsin K is a target for the treatment of diseases associated with high bone turnover. Cathepsin K is mainly expressed in osteoclasts and responsible for the destruction of the proteinaceous components of the bone matrix. We designed various fluorescent activity-based probes (ABPs) and their precursors that bind to and inactivate cathepsin K. ABP 25 exhibited extraordinary potency (kinac/Ki = 35,300 M-1s-1) and selectivity for human cathepsin K. Crystal structures of cathepsin K in complex with ABP 25 and its nonfluorescent precursor 21 were determined to characterize the binding mode of this new type of acrylamide-based Michael acceptor with the particular orientation of the dibenzylamine moiety to the primed subsite region. The cyanine-5 containing probe 25 allowed for sensitive detection of cathepsin K, selective visualization in complex proteomes, and live cell imaging of a human osteosarcoma cell line, underlining its applicability in a pathophysiological environment.

Spatial expression pattern of serine proteases in the blood fluke Schistosoma mansoni determined by fluorescence RNA in situ hybridization.

BACKGROUND: The blood flukes of genus Schistosoma are the causative agent of schistosomiasis, a parasitic disease that infects more than 200 million people worldwide. Proteases of schistosomes are involved in critical steps of host-parasite interactions and are promising therapeutic targets. We recently identified and characterized a group of S1 family Schistosoma mansoni serine proteases, including SmSP1 to SmSP5. Expression levels of some SmSPs in S. mansoni are low, and by standard genome sequencing technologies they are marginally detectable at the method threshold levels. Here, we report their spatial gene expression patterns in adult S. mansoni by the high-sensitivity localization assay. METHODOLOGY: Highly sensitive fluorescence in situ RNA hybridization (FISH) was modified and used for the localization of mRNAs encoding individual SmSP proteases (including low-expressed SmSPs) in tissues of adult worms. High sensitivity was obtained due to specifically prepared tissue and probes in combination with the employment of a signal amplification approach. The assay method was validated by detecting the expression patterns of a set of relevant reference genes including SmCB1, SmPOP, SmTSP-2, and Sm29 with localization formerly determined by other techniques. RESULTS: FISH analysis revealed interesting expression patterns of SmSPs distributed in multiple tissues of S. mansoni adults. The expression patterns of individual SmSPs were distinct but in part overlapping and were consistent with existing transcriptome sequencing data. The exception were genes with significantly low expression, which were also localized in tissues where they had not previously been detected by RNA sequencing methods. In general, SmSPs were found in various tissues including reproductive organs, parenchymal cells, esophagus, and the tegumental surface. CONCLUSIONS: The FISH-based assay provided spatial information about the expression of five SmSPs in adult S. mansoni females and males. This highly sensitive method allowed visualization of low-abundantly expressed genes that are below the detection limits of standard in situ hybridization or by RNA sequencing. Thus, this technical approach turned out to be suitable for sensitive localization studies and may also be applicable for other trematodes. The results suggest that SmSPs may play roles in diverse processes of the parasite. Certain SmSPs expressed at the surface may be involved in host-parasite interactions.

Advanced Real-Time Process Analytics for Multistep Synthesis in Continuous Flow*.

In multistep continuous flow chemistry, studying complex reaction mixtures in real time is a significant challenge, but provides an opportunity to enhance reaction understanding and control. We report the integration of four complementary process analytical technology tools (NMR, UV/Vis, IR and UHPLC) in the multistep synthesis of an active pharmaceutical ingredient, mesalazine. This synthetic route exploits flow processing for nitration, high temperature hydrolysis and hydrogenation reactions, as well as three inline separations. Advanced data analysis models were developed (indirect hard modeling, deep learning and partial least squares regression), to quantify the desired products, intermediates and impurities in real time, at multiple points along the synthetic pathway. The capabilities of the system have been demonstrated by operating both steady state and dynamic experiments and represents a significant step forward in data-driven continuous flow synthesis.