Practical QSP application from the preclinical phase to enhance the probability of clinical success: Insights from case studies in oncology.

Quantitative Systems Pharmacology (QSP) has emerged as a promising modeling and simulation (M&S) approach in drug development, with potential to improve clinical success rates. While conventional M&S has significantly contributed to quantitative understanding in late preclinical and clinical phases, it falls short in explaining unexpected phenomena and testing hypotheses in the early research phase. QSP presents a solution to these limitations. To harness the full potential of QSP in early preclinical stages, preclinical modelers who are familiar with conventional M&S need to update their understanding of the differences between conventional M&S and QSP. This review focuses on QSP applications during the preclinical stage, citing case examples and sharing our experiences in oncology. We emphasize the critical role of QSP in increasing the probability of success for clinical proof of concept (PoC) when applied from the early preclinical stage. Enhancing the quality of both hypotheses and QSP models from early preclinical stage is of critical importance. Once a QSP model achieves credibility, it facilitates predictions of clinical responses and potential biomarkers. We propose that sequential QSP applications from preclinical stages can improve success rates of clinical PoC, and emphasize the importance of refining both hypotheses and QSP models throughout the process.

Nannostelium ampullaceum gen. et sp. nov., a tiny new member of the protosteloid amoeba of the Cavosteliida (Variosea, Amoebozoa).

Protosteloid amoebae are slime molds characterized by simple sporocarp comprised only of a stalk and typically single spore. Recent molecular phylogenetic studies showed that protosteloid amoebae are scattered among the Amoebozoa, but most of them belongs to the Variosea. The Cavosteliida is one of the largest protosteloid group of the Variosea. We have established novel protosteloid amoeba strain YIP-63 from the fruiting body of jelly fugus Auricularia sp. The strain YIP-63 is unique to have a tiny sporocarp and amoeba comparing to the other protosteloid species, and trophic amoebae forming both round and branched shape. The molecular phylogenetic analyses based on 18S rRNA gene suggest that YIP-63 represents a novel lineage in the Cavosteliida. Therefore, we propose the new genus and new species for the strain YIP-63 as Nannostelium ampullaceum gen. et sp. nov. We provide morphological and molecular data on this novel protosteloid amoeba.

Taxonomy of a New Parasitic Euglenid, Euglenaformis parasitica sp. nov. (Euglenales, Euglenaceae) in Ostracods and Rhabdocoels.

Parasitic euglenids have rarely been studied. We found parasitic euglenids in two species of ostracods (Cyprinotus cassidula, Dolerocypris sinensis) and two species of rhabdocoels (Mesostoma lingua, Microdalyellia armigera) in a rice field. These parasites grew and proliferated inside the host body. These parasites had pellicle strips, one emergent flagellum, and a red stigma, but no chloroplasts, and showed euglenoid movement. Inside the living host, they did not have emergent flagella and moved only by euglenoid movement, but when the host died or the parasites were isolated from the host, they extended their flagella and switched to swimming movement. We conclude that the parasites found in the four hosts that we examined are of the same species, considering the morphological characteristics and identities in the nSSU and nLSU rDNA sequences of those parasites. Molecular phylogenetic analysis showed that the parasite formed a clade with the free-living photoautotrophic species of Euglenaformis, with moderate statistical support. Therefore, the parasite is a secondary osmotroph derived from a photoautotrophic ancestor. Based on the results of morphological observation and molecular phylogenetic analysis, we propose a new species of parasitic euglenid, Euglenaformis parasitica sp. nov.

Re-examination of a rare protosteloid amoeba Schizoplasmodiopsis micropunctata, and the revision of Tychosporium (Cavosteliida, Variosea, Amoebozoa).

The genus Schizoplasmodiopsis is one of the most morphologically diverse groups among the class Variosea. Recent phylogenetic studies suggest that Schizoplasmodiopsis is polyphyletic, but there are few taxonomic studies of this genus. We established S. micropunctata strain YIP-40, observed in detail its of morphology and lifecycle, and conducted a phylogenetic analysis. The phylogenetic analysis revealed that S. micropunctata was sister to Tychosporium acutostipes. Scanning electron microscopy showed S. micropunctata had a non-deciduous hilum structure that is unique to Tychosporium. The morphology of amoebae, mitotic behavior, and prespore cells of S. micropunctata also supported the close relationship to Tychosporium. We propose to transfer S. micropunctata to Tychosporium and emend the generic concept of Tychosporium to include this species.

Frequency Error Compensation of Unsynchronized Bistatic CW- MIMO Radar for Multiple Human-Body Localization.

This article presents and experimentally evaluates a frequency error elimination technique suitable for unsynchronized bistatic Multiple-Input Multiple-Output (MIMO) radar for human-body detection. First, a mathematical expression of human-body localization using bistatic MIMO radar is presented. Then the direct path is used to eliminate the phase error created by the frequency difference between the transmitter and receiver. A new Doppler-shifted component of the MIMO channel without phase error is derived, and the locations of the multiple targets are calculated by the 2-dimensional MUltiple SIgnal Classification (MUSIC) method. Next, the results of simulations that examine frequency error versus power ratios are discussed to illustrate the effectiveness of the proposed method. An experiment is carried out in an indoor multipath-rich environment. To emulate the unsynchronized condition, the transmitter and receiver use independent Signal Generators (SGs). One to six targets are tested. The experiments demonstrate that our unsynchronized radar system can identify the locations of multiple targets with high accuracy.

Taxonomic study of a new green alga, Annulotesta cochlephila gen. et sp. nov. (Kornmanniaceae, Ulvales, Ulvophyceae), growing on the shells of door snails.

Some algae are known to grow on shellfish shells. Most of these have been reported in aquatic environments. The species specificity for substrate shells varies, and some algae grow only on the shells of a certain species of shellfish, such as Pseudocladophora conchopheria (Cladophorales, Ulvophyceae) on Lunella coreensis (Trochida, Gastropoda). There are very few reports of algae that grow on land snails. In this study, we discovered green algae growing on the shells of six species of door snails (Clausiliidae) from nine localities in Japan. These green algae formed a green mat composed of thalli embedded in the extracellular matrix. The thallus was composed of aggregated oval cells and peripheral branched filaments. The cells possessed a single parietal chloroplast with a pyrenoid surrounded by two starch sheaths and transversed by a thylakoid. Oil droplets in the cell and ring-like structures on the cell wall surface were frequently observed. The 18S rDNA sequences of all shell-attached algae on different clausiliid species from different localities were almost identical and formed a new clade in the family Kornmanniaceae (Ulvales, Ulvophyceae). No other algae forming visible colonies on the clausiliid shell were found. These findings indicate the presence of specificity between the alga and clausiliid shells. Based on the results of morphological observation and molecular phylogenetic analysis, we propose a new genus and new species of shell-attached green alga, Annulotesta cochlephila.

Revisiting the isolation source after half a century: Emericellopsis mirabilis on a yellow-green alga.

Fungi-algae interactions, such as lichen-forming fungi and parasitic chytrids on phytoplankton, are common in ecosystems. In contrast, interactions between filamentous fungi and soil algae that can be observed with the naked eye have been given little attention and remain unexplored. Here, we report a fungus that was associated with a visible symptom of dead algae on a soil surface in Sugadaira-kogen, Nagano, central Japan. Acremonium-like conidiophores were growing on vesicles and dead bodies of a yellow-green alga, Botrydium granulatum. The fungus was identified as Emericellopsis mirabilis based on its morphology by microscopic observation, phylogenetic analysis, and the similarity of the isolation substrate with the first description of the species. Co-culture experiments showed a filamentous cell differentiation of the alga by the fungus, but no harmful or beneficial effects on algal growth. Therefore, we speculate that E. mirabilis is a facultative parasite of B. granulatum under natural conditions.

Sex-specific Positioning of the Mating Structure in Scale-bearing Gametes of Monostroma angicava and Collinsiella cava (Ulvophyceae, Chlorophyta): A Possible Widespread Difference between Male and Female Gametes.

The gametes of chlorophytes can be divided into two morphological types (types α and ß) based on the position of the mating structure relative to the flagella and eyespot. To elucidate the relationship between the morphological types and the sexes, we studied spatial relationships between the flagellar apparatus-eyespot-mating structures in biflagellate male and female gametes and their fate after fertilization in the anisogamous (Monostroma angicava) and the slightly anisogamous species (Collinsiella cava) using field emission scanning electron microscopy and transmission electron microscopy. The smaller male and larger female gametes of M. angicava had two basal bodies arranged at a 180° angle and the cell surface coated with square-shaped body scales, except for the flagella and mating structures. The mating structure of the female gamete was located on the same side of the flagellar beat plane as the eyespot (type ß), whereas that of the male gamete was located on the opposite side (type α). This mating structure arrangement was also confirmed in C. cava. The initial fusion when male and female gametes were mixed involved the mating structures. In a fusing pair of gametes, each flagellum of one gamete lay alongside one flagellum of the other gamete. As fusion proceeded, the gamete pair transformed into a quadriflagellate planozygote, in which the four basal bodies were arranged in a cruciate pattern. The eyespots were positioned side-by-side on the same side of the cell. These results suggest that the two morphological types of gametes are intimately correlated with the particular sexes.

Partial silencing of fucosyltransferase 8 gene expression inhibits proliferation of Ishikawa cells, a cell line of endometrial cancer.

Endometrial cancer is the most common gynecologic malignancy and is associated with increased morbidity each year, including young people. However, its mechanisms of proliferation and progression are not fully elucidated. It is well known that abnormal glycosylation is involved in oncogenesis, and fucosylation is one of the most important types of glycosylation. In particular, fucosyltransferase 8 (FUT8) is the only FUT responsible for α1, 6-linked fucosylation (core fucosylation), and it is involved in various physiological as well as pathophysiological processes, including cancer biology. Therefore, we aimed to identify the expression of FUT8 in endometrial endometrioid carcinoma and investigate the effect of the partial silencing of the FUT8 gene on the cell proliferation of Ishikawa cells, an epithelial-like endometrial cancer cell line. Quantitative real-time PCR analysis showed that FUT8 gene expression was significantly elevated in the endometrial endometrioid carcinoma, compared to the normal endometrium. The immunostaining of FUT8 and Ulex europaeus Agglutinin 1 (UEA-1), a kind of lectin family specifically binding to fucose, was detected endometrial endometrioid carcinoma. The proliferation assay showed FUT8 partial knockdown by transfection of siRNA significantly suppressed the proliferation of Ishikawa cells, concomitant with the upregulation in the gene expressions associated with the interesting pathways associated with de-ubiquitination, aspirin trigger, mesenchymal-epithelial transition (MET) et al. It was suggested that the core fucosylation brought about by FUT8 might be involved in the proliferation of endometrial endometrioid carcinoma cells.

Microtubule stabilizer reveals requirement of Ca2+-dependent conformational changes of microtubules for rapid coiling of haptonema in haptophyte algae.

A haptonema is an elongated microtubule-based motile organelle uniquely present in haptophytes. The most notable and rapid movement of a haptonema is 'coiling', which occurs within a few milliseconds following mechanical stimulation in an unknown motor-independent mechanism. Here, we analyzed the coiling process in detail by high-speed filming and showed that haptonema coiling was initiated by left-handed twisting of the haptonema, followed by writhing to form a helix from the distal tip. On recovery from a mechanical stimulus, the helix slowly uncoiled from the proximal region. Electron microscopy showed that the seven microtubules in a haptonema were arranged mostly in parallel but that one of the microtubules often wound around the others in the extended state. A microtubule stabilizer, paclitaxel, inhibited coiling and induced right-handed twisting of the haptonema in the absence of Ca2+, suggesting changes in the mechanical properties of microtubules. Addition of Ca2+ resulted in the conversion of haptonematal twist into the planar bends near the proximal region. These results indicate that switching microtubule conformation, possibly with the aid of Ca2+-binding microtubule-associated proteins is responsible for rapid haptonematal coiling.

Viridiuvalis adhaerens gen. et sp. nov., a novel colony-forming chlorarachniophyte.

A new chlorarachniophyte, Viridiuvalis adhaerens gen. et sp. nov. was isolated from the mucus on a coral reef from Zanpa Beach, Okinawa, Japan. The main vegetative stage of V. adhaerens consisted of unicellular coccoid cells with cell walls, although sarcinoid colonies and uniflagellate zoospores were also observed. V. adhaerens had chloroplasts with nucleomorphs and pyrenoids that were completely embedded in the chloroplast. A deep plate-like invagination of the periplastidal compartment (PPC) almost partitioned the pyrenoid and chloroplast components, which were surrounded by two membranes. The nucleomorph was positioned in the base of the invagination of the PPC. Molecular phylogenetic analyses using rRNA genes showed that V. adhaerens branched as a sister lineage of the Amorphochlora clade. The sarcinoid colony, pyrenoid embedded in the chloroplast, and nucleomorph located at the base of the deep invagination of the PPC have not been reported in other chlorarachniophytes. Based on these morphological and ultrastructural characteristics and the results of the molecular phylogenetic analyses, we propose V. adhaerens as a new genus and species of chlorarachniophyte.

A Clinical Cassette Dosing Study for Evaluating the Contribution of Hepatic OATPs and CYP3A to Drug-Drug Interactions.

PURPOSE: To demonstrate the relative importance of organic anion-transporting polypeptides (OATPs) and cytochrome P450 3A (CYP3A) in the hepatic elimination of substrate drugs. METHODS: A cocktail of subtherapeutic doses of bosentan, repaglinide, clarithromycin, darunavir, simeprevir, and midazolam (CYP3A probe) was administered orally to eight healthy volunteers. Rifampicin (OATP inhibitor; 600 mg, p.o.) and itraconazole (CYP3A inhibitor; 200 mg, i.v.) were coadministered with the cocktail in the second and third phases, respectively. Based on the extended clearance concept, in vivo ß values (fraction of metabolism plus biliary excretion among all the intracellular fates of drugs including basolateral efflux) and Rdif values (ratio of diffusional uptake to active uptake) were estimated. RESULTS: Rifampicin increased plasma AUCs of bosentan (×3.2), repaglinide (×1.9), clarithromycin (×1.9) and simeprevir (×7.2). Itraconazole increased those of clarithromycin (×2.3), simeprevir (×2.2) and midazolam (×3.7), which had relatively small ß values. The plasma AUC of bosentan (with relatively large ß and small Rdif) was dominated by OATP-mediated uptake. The AUC of simeprevir was also dominated by OATP-mediated uptake because of its small Rdif value. CONCLUSIONS: The DDI study clarified the rate-determining processes of OATP/CYP3A substrates. Our analyses provide valuable information for predicting complex drug-drug interactions involving multiple processes.

Investigation of Glycochenodeoxycholate Sulfate and Chenodeoxycholate Glucuronide as Surrogate Endogenous Probes for Drug Interaction Studies of OATP1B1 and OATP1B3 in Healthy Japanese Volunteers.

PURPOSE: To assess the use of glycochenodeoxycholate-3-sulfate (GCDCA-S) and chenodeoxycholate 3- or 24-glucuronide (CDCA-3G or -24G) as surrogate endogenous substrates in the investigation of drug interactions involving OATP1B1 and OATP1B3. METHODS: Uptake of GCDCA-S and CDCA-24G was examined in HEK293 cells transfected with cDNA for OATP1B1, OATP1B3, and NTCP and in cryopreserved human hepatocytes. Plasma concentrations of bile acids and their metabolites (GCDCA-S, CDCA-3G, and CDCA-24G) were determined by LC-MS/MS in eight healthy volunteers with or without administration of rifampicin (600 mg, po). RESULTS: GCDCA-S and CDCA-24G were substrates for OATP1B1, OATP1B3, and NTCP. The uptake of [3H]atorvastatin, GCDCA-S, and CDCA-24G by human hepatocytes was significantly inhibited by both rifampicin and pioglitazone, whereas that of taurocholate was inhibited only by pioglitazone. Rifampicin elevated plasma concentrations of GCDCA-S more than those of other bile acids. The area under the plasma concentration-time curve for GCDCA-S was 20.3 times higher in rifampicin-treated samples. CDCA-24G could be detected only in plasma from the rifampicin-treatment phase, and CDCA-3G was undetectable in both phases. CONCLUSIONS: We identified GCDCA-S and CDCA-24G as substrates of NTCP, OATP1B1, and OATP1B3. GCDCA-S is a surrogate endogenous probe for the assessment of drug interactions involving hepatic OATP1B1 and OATP1B3.

The effect of oil and water-soluble contrast medium in hysterosalpingography on thyroid function.

Hysterosalpingography (HSG) using iodinated contrast medium is X-ray diagnostic test that examines the inside of the uterus and fallopian tubes in infertile women. In this study, we compared thyroid function (thyroid stimulating hormone: TSH and free-T4: FT4 levels) after HSG with an oil-soluble contrast medium (OSCM) and a water-soluble contrast medium (WSCM). One hundred and sixty-four and 94 patients with normal thyroid function received HSG with OSCM and WSCM, respectively. Approximately 25% of the women in the OSCM group developed subclinical hypothyroidism (SCH), whereas only less than 10% of the patients in the WSCM group developed SCH. Our data clearly indicate that WSCM is safe for thyroid function in women who plan to get pregnant.

Mano-videoendoscopic assessment in the evaluation of the pharyngeal contraction and upper esophageal sphincter function in dysphagic patients.

OBJECTIVE: Mano-videoendoscopy (MVE) is a manometry technique with endoscopic confirmation of the pressure catheter. This study aimed to investigate the possibility of replacing a videofluorographic swallowing study (VFSS) with MVE for the precise evaluation of the pharyngeal contraction and the upper esophageal sphincter (UES) function. METHODS: The data from 69 patients with dysphagia were retrospectively reviewed. All of the patients underwent both MVE and a VFSS for the evaluation of dysphagia. Manometry was performed with a transnasally inserted catheter (2.6-mm outer diameter and 4 pressure sensors) under endoscopic observation. The sensors were kept at the tongue base, upper pyriform sinus, apex of the pyriform sinus, and UES. We evaluated the pharyngeal contraction and UES function fluorographically and statistically compared the manometric parameters. RESULTS: The fluorographic pharyngeal contraction was diagnosed as good in 28 patients and poor in 41 patients. The UES opening was diagnosed as good in 44 patients and poor in 25 patients. The highest pressure values at the tongue base (sensor 1), upper pyriform sinus (sensor 2), and apex of the pyriform sinus (sensor 3) were significantly larger in the good contraction group than in the poor contraction group. A stepwise logistic regression test revealed that the peak pressure of sensor 2 (upper pyriform sinus) was a robust predictor of fluorographic pharyngeal contraction, and the cut-off level for good fluorographic pharyngeal contraction was >81.5mmHg (specificity, 0.929; sensitivity, 0.870; area under the curve, 0.923). The nadir pressure, pressure drop, and pressure rise in the UES were significantly correlated with the fluorographic UES opening. A stepwise logistic regression test revealed that the pressure drop-the gap between the resting pressure and the nadir of the UES pressure-was a robust predictor of fluorographic UES opening, and the cut-off level to anticipate good fluorographic opening was ≥33.5mmHg (specificity, 0.853; sensitivity, 0.759). CONCLUSION: MVE can supplement the information obtained regarding the pharyngeal contraction and UES function, and overcomes the drawbacks of a videoendoscopic swallowing study (VESS).

A New Deep-branching Stramenopile, Platysulcus tardus gen. nov., sp. nov.

A novel free-living heterotrophic stramenopile, Platysulcus tardus gen. nov., sp. nov. was isolated from sedimented detritus on a seaweed collected near the Ngeruktabel Island, Palau. P. tardus is a gliding flagellate with tubular mastigonemes on the anterior short flagellum and a wide, shallow ventral furrow. Although the flagellar apparatus of P. tardus is typical of stramenopiles, it shows novel ultrastructural combinations that are not applied to any groups of heterotrophic stramenopiles. Phylogenetic analysis using SSU rRNA genes revealed that P. tardus formed a clade with stramenopiles with high support. However, P. tardus did not form a subclade with any species or environmental sequences within the stramenopiles, and no close relative was suggested by the phylogenetic analysis. Therefore, we concluded that P. tardus should be treated as a new genus and species of stramenopiles and have proposed a new family, Platysulcidae fam. nov., for this phylogenetically distinct organism.

Can mano-videoendoscopy substitute for videofluorography in evaluation of upper esophageal sphincter function?

CONCLUSIONS: Mano-videoendoscopy (MVE), a manometry technique with endoscopic confirmation of the pressure catheter, can supplement the information on upper esophageal sphincter (UES) function, and overcomes the drawbacks of videoendoscopic swallowing study (VESS). OBJECTIVES: This study aimed to investigate the possibility of replacing videofluorographic swallowing study (VFSS) with MVE, as a test to precisely evaluate UES function. METHODS: Data from 52 patients with dysphagia were retrospectively reviewed. All patients underwent both MVE and VFSS for evaluation of dysphagia. The manometry was performed with a transnasally inserted catheter (2.6 mm outer diameter and four pressure sensors) under endoscopic observation. The sensors were kept at the tongue base, upper pyriform sinus, apex of pyriform sinus, and UES. We statistically compared the manometric parameters of UES relaxation with fluorographic UES opening. RESULTS: Fluorographic UES opening was diagnosed as good in 34 patients and poor in 18 patients. The nadir pressure, pressure drop, and pressure rise in the UES had significant correlation on the fluorographic UES opening. Stepwise logistic regression test revealed that pressure drop, the gap between the resting pressure and the nadir of UES pressure, was a robust parameter for predicting fluorographic UES opening, and the cut-off level to anticipate good fluorographic opening was ≥ 33.5 mmHg (specificity, 0.853; sensitivity, 0.759).

Taxonomic study of a new eustigmatophycean alga, Vacuoliviride crystalliferum gen. et sp. nov.

This study investigated the taxonomic affiliation of the algal strain nak-9, which has been reported to absorb radioactive cesium with high efficiency, using light and electron microscopy, and molecular phylogenetic analysis based on 18S ribosomal RNA gene (rDNA) sequences. This alga is spherical and coccoid, with a smooth cell wall, large vacuole, crystalline structure, reddish globule, and refractile granules (lamellate vesicles). The cells possess one to several greenish parietal chloroplasts with a bulging pyrenoid surrounded by lamellate vesicles. The chloroplasts include orderly thylakoid lamellae but no girdle lamella. Molecular phylogenetic analysis suggests that strain nak-9 is a member of the eustigmatophycean clade, which includes Goniochloris, Pseudostaurastrum, and Trachydiscus. On the basis of these results, we propose that strain nak-9 (NIES-2860) comprises a new species and new genus of the Eustigmatophyceae, Vacuoliviride crystalliferum gen. et sp. nov.

Molecular diversity of endosymbiotic Nephroselmis (Nephroselmidophyceae) in Hatena arenicola (Katablepharidophycota).

Hatena arenicola (Katablepharidophycota) is a single-celled eukaryote that temporarily possesses a chlorophyte alga of the genus Nephroselmis as an intracellular symbiont. In the present study, we investigated the molecular diversity of the endosymbiont Nephroselmis in a natural population of the host H. arenicola. We sequenced the host's 18S rRNA gene and the endosymbiont's plastid-encoded 16S rRNA gene. The results indicated that almost identical strains of the host harbored at least three distinct strains of the algal endosymbiont affiliated to the clade Nephroselmis rotunda. This finding supports our previous hypothesis that H. arenicola and its symbiotic alga are in an early stage of secondary endosymbiosis.