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Abstract Background The neck imaging reporting and data system (NI-RADS) is a structured reporting algorithm linked with further patient management recommendations. This study was conducted to assess the overall and time point predictive value of the NI-RADS in laryngeal and oral cavity squamous cell carcinoma (SCC) using contrast-enhanced magnetic resonance imaging (CEMRI) and contrast-enhanced computed tomography (CECT). Results The rate of tumor recurrence was statistically different among the NI-RADS 1-3 categories with recurrence trend for higher NI-RADS scores. The overall negative predictive value (NPV) of the NI-RADS 1 and 2 were 94.3%, 74.3% respectively, and the positive predictive value (PPV) of the NI-RADS 3 was 80.8%. The overall recurrence rate of NI-RADS 3 was higher in oral cavity SCC (87.5%) compared to the laryngeal SCC (70%). The PPV of overall NI-RADS 3 in the follow-up scans (77.8%) was higher than in the first scan (70.6%). The odd ratio of tumor recurrence in NI-RADS 3 primary lesion was 19.6. Conclusion The predictive value of NI-RADS was significantly different among its categories. Increasing NI-RADS score is associated with increased recurrence among the treated laryngeal and oral cavity SCC. The morphological and enhancement lexicon features equally assign the NI-RADS 3 score.

Abstract Background To evaluate the inter- and intrareader agreement and reproducibility of the NI-RADS scoring system and lexicon with contrast-enhanced computed tomography (CECT) and contrast-enhanced magnetic resonance imaging (CEMRI). Methods This retrospective study included 97 CECT and CEMRI scans from 58 treated cases of head and neck squamous cell carcinoma (HNSCC) after the exclusion of head and neck cancers (HNCs) other than SCC and noncontrast and poor quality CT and MRI scans, with a total of 111 primary targets and 124 lymph node (LN) targets. Two experienced readers independently scored the likelihood of residual/recurrence for these targets based on the NI-RADS criteria and filled in report templates for NI-RADS lexicon diagnostic features. Inter- and intraobserver reproducibility was assessed with Cohen’s kappa, and the percent agreement was calculated. Results Almost perfect interreader agreement was found for the final NI-RADS category of the primary lesions and LNs, with K = 0.808 and 0.806, respectively. Better agreement was found for CT than for MRI (K = 0.843 and 0.77, respectively, P value 0.001). There was almost perfect agreement for excluding tissue enhancement (K = 0.826, 95% CI = 0.658–0.993, P value 0.001), with a percent agreement of 96.4%, and substantial agreement for discrete nodular and diffuse mucosal enhancement (K = 0.826, 95% CI = 0.658–0.993, P value 0.001), with a percent agreement of 96.4%. There was fair agreement for focal mucosal nonmass and deep ill-defined enhancement. The intrareader agreement was almost perfect for most of the rated features (K ranging from 0.802 to 1), with the exception of enlarging discrete nodule/mass and focal mucosal nonmass-like enhancement, which had substantial intraobserver agreement (K ranging from 0.768 to 0.786). Conclusion The individual features of NI-RADS show variable degrees of confidence; however, the overall NI-RADS category was not significantly affected.

Abstract Background Recovery of upper extremity (UE) motor function after stroke is variable from one to another due to heterogeneity of stroke pathology. Structural and biochemical magnetic resonance imaging of the primary motor cortex (M1) have been used to document reorganization of neural activity after stroke. Objective To assess cortical biochemical and structural causes of delayed recovery of UE motor function impairment in chronic subcortical ischemic stroke patients. Methodology A cross-sectional study with fifty patients were enrolled: thirty patients with chronic (> 6 months) subcortical ischemic stroke suffering from persistent UE motor function impairment (not improved group) and twenty patients with chronic subcortical ischemic stroke and improved UE motor function (improved group). We recruited a group of (16) age-matched healthy subjects. Single voxel proton magnetic resonance spectroscopy (1H-MRS) was performed to measure n-acetylaspartate (NAA) and glutamate+glutamine (Glx) ratios relative to creatine (Cr) in the precentral gyrus which represent M1of hand area in both ipsilesional and contralesional hemispheres. Brain magnetic resonance imaging (MRI) to measure precentral gyral thickness is representing the M1of hand area. UE motor function assessment is using the Fugl Meyer Assessment (FMA-UE) Scale. Results The current study found that ipslesional cortical thickness was significantly lower than contralesional cortical thickness among all stroke patients. Our study found that ipsilesional NAA/Cr ratio was lower than contralesional NAA/Cr among stroke patients. UE and hand motor function by FMA-UE showed highly statistically significant correlation with ipsilesional cortical thickness and ipsilesional NAA/Cr ratio, more powerful with NAA/Cr ratio. Conclusion We concluded that persistent motor impairment in individuals with chronic subcortical stroke may be at least in part related to ipsilesional structural and biochemical changes in motor areas remote from infarction in form of decreased cortical thickness and NAA/Cr ratio which had the strongest relationship with that impairment.

Abstract Background Olfaction is critically important for a good quality of life and incorporated in many physiological domains such as attention, emotion, memory, and airflow motor control. Olfactory researches have been expanded in the last decade due to close relation between the olfactory disorders and different brain diseases. Diagnosis of anosmia and hyposmia are based on history, smell tests, and physical examination which rely on the patient’s response without an objective measure of impairment. This study assessed the value of volumetry and DTI parameters as objective measurements for olfactory dysfunction. Fourteen patients with chronic sinonasal polyposis for at least 6 months were included in this study; all of them underwent tailored MRI examination including volumetry and DTI for olfactory bulbs and tracts. The results were compared to the same number of age and sex-matched healthy control group. Results The study results showed that olfactory bulb and tract (OB/T) volume, FA and ADC could distinguish between patients and healthy controls. Statistically significant differences were noticed between the FA & ADC values of patient and control groups (p < 0.05) and a highly significant one was noticed as regarding the OT volume (p < 0.001). Conclusion MR volumetry and DTI parameters can be used as objective measurements for the olfactory dysfunction for patients with chronic sinonasal polyposis.

Objective: To determine if there was an increase in the rate of cases presenting with rhino-orbital-cerebral mucormycosis (ROCM) to a tertiary care center during the first wave of the coronavirus disease 2019 (COVID-19) pandemic and the characteristics of the presenting cases.Methods: Retrospective observational study reviewing ROCM cases presenting from March 25 until September 25, 2020. Cases fulfilling the clinical, radiological, and pathological/microbiological criteria for diagnosis with ROCM were included. The number of cases presenting during the designated interval, their COVID-19 status, comorbidities, and clinical presentation were analyzed. The number of cases during the corresponding interval in the previous 3 years was used as reference to detect if there was a recent spike.Results: Of the 12 ROCM cases identified, 5 had a concurrent positive reverse transcription PCR (RT-PCR) test result for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), 1 had a prior positive result, and 6 did not have concurrent nor prior positive test results. Nine of the 12 cases had poorly controlled diabetes mellitus, and 2 cases had a hematological malignancy. All cases had orbital invasion, and eight cases had cerebral invasion. The number of cases identified during the interval is much higher than the numbers presenting in the prior 3 years during equivalent intervals (range, one to two cases) than those reported in the literature in different settings in the pre-pandemic era.Conclusions: There is an increased rate of ROCM cases presenting to our center during the first wave of the COVID-19 pandemic. This is a preliminary report, and further studies are needed to corroborate the findings and explain possible underlying links.

Purpose: In pediatric population with SNHL, a radiological presurgical mapping exam is usually performed, purpose of this study is to improve the inter-rater agreement giving different imaging modalities using MDCT and MRI to facilitate identifying patients in need for surgical modification before cochlear implant. Methods and material: 120 pediatric patients ranging between 1 and 12 years with an average age of 5 years diagnosed with congenital or acquired SNHL and requiring cochlear implant (CI) after proper surgical selection were included in the study. All patients underwent combined 64 detector MDCT using pediatric low dose protocol with sagittal, Stenvers views in addition to standard axial and coronal plane reconstruction from single acquisition volume. In the same session, 3D DRIVE MRI axial plane reformatted into sagittal plane for the IAC and inner ear as well as axial T2WI/FLAIR for the whole brain. Two different head and neck radiologists blindedly reported the inner ear, cochlear nerve development, temporal bone anatomy, operative window, as well as normal variants that could hinder implantation as well as causes of central hearing loss. Findings were compared with operative findings. Results: Patients were categorized into four groups; according to the capability of electrode implant, modification of surgical techniques and expected response with high intra-observer was noted with a PABAK value of 0.96 for observer A and of 0.93 for observer B. Lower inter observer agreement was observed on an individual usage of radiological techniques for reaching diagnosis (conventional CT, MRI, or Stenvers/sagittal oblique techniques alone) with a PABAK value of 0.93, 0.78 and 0.73, respectively. A higher inter observer agreement was met on combining all these radiological modalities together (PABAK value of 0.96). Conclusion: Stenvers view efficiently makes pre-surgical mapping for the intra-cochlear electrode pathway by identifying the cochlear turns, round and oval window, vestibule and SCC as well as facial nerve canal in one view helping the surgeon in optimizing the technique. MRI identifies the fibrous labyrinthitis ossificans not detected by CT and evaluates the cochlear nerve development and integrity of central auditory pathway. Combined MDCT/MRI imaging protocol helps the clinician in proper patient categorization, optimize their surgical approach and the type of electrode. Clinical relevance/application: SNHL is a malfunction of the inner ear, vestibulocochlear nerve or central auditory pathway. Imaging helps to establish treatment regimens that improve auditory function.

A novel trimeric monoterpenoid indole alkaloid, vincarostine A (1) consisting of an aspidosperma-iboga-aspidosperma type skeleton, was isolated from the whole plant of Catharanthus roseus. The structure including absolute stereochemistry was elucidated on the basis of 2D NMR data and CD spectrum. Vincarostine A (1) showed anti-malarial activity., (© 2024. The Author(s) under exclusive licence to The Japanese Society of Pharmacognosy.)

The call for second generation malaria vaccines needs not only the identification of novel candidate antigens or adjuvants but also a better understanding of immune responses and the underlying protective processes. Plasmodium parasites have evolved a range of strategies to manipulate the host immune system to guarantee survival and establish parasitism. These immune evasion strategies hamper efforts to develop effective malaria vaccines. In the case of a malaria vaccine targeting the N-terminal domain of P. falciparum serine repeat antigen 5 (SE36), now in clinical trials, we observed reduced responsiveness (lowered immunogenicity) which may be attributed to immune tolerance/immune suppression. Here, immunogenicity data and insights into the immune responses to SE36 antigen from epidemiological studies and clinical trials are summarized. Documenting these observations is important to help identify gaps for SE36 continued development and engender hope that highly effective blood-stage/multi-stage vaccines can be achieved., Competing Interests: Declaration of Competing Interest TH is the inventor of BK-SE36; TH, KJI, and TT are inventors of BK-SE36/CpG. NP and TH are both supported by a research fund from Nobelpharma Co., Ltd. (NPC), the clinical trial sponsor of Burkina Faso trials. These involvements did not influence the writing of this review and the decision to submit the article for publication., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Objectives: To propose an automated fast cochlear segmentation, length, and volume estimation method from clinical 3D multimodal images which has a potential role in the choice of cochlear implant type, surgery planning, and robotic surgeries., Methods: Two datasets from different countries were used. These datasets include 219 clinical 3D images of cochlea from 3 modalities: CT, CBCT, and MR. The datasets include different ages, genders, and types of cochlear implants. We propose an atlas-model-based method for cochlear segmentation and measurement based on high-resolution μ CT model and A -value. The method was evaluated using 3D landmarks located by two experts., Results: The average error was 0.61 ± 0.22 mm and the average time required to process an image was 5.21 ± 0.93 seconds ( P <0.001). The volume of the cochlea ranged from 73.96 mm 3 to 106.97 mm 3 , the cochlear length ranged from 36.69 to 45.91 mm at the lateral wall and from 29.12 to 39.05 mm at the organ of Corti., Discussion: We propose a method that produces nine different automated measurements of the cochlea: volume of scala tympani, volume of scala vestibuli, central lengths of the two scalae, the scala tympani lateral wall length, and the organ of Corti length in addition to three measurements related to A -value., Conclusion: This automatic cochlear image segmentation and analysis method can help clinician process multimodal cochlear images in approximately 5 seconds using a simple computer. The proposed method is publicly available for free download as an extension for 3D Slicer software.

Ceramicines are a series of limonoids which were isolated from the barks of Malaysian Chisocheton ceramicus (Meliaceae), and were known to show various biological activity. Six new limonoids, ceramicines U-Z (1-6), with a cyclopentanone[α]phenanthrene ring system with a β-furyl ring at C-17 were isolated from the barks of C. ceramicus. Their structures were determined on the basis of the 1D and 2D NMR analyses, and their absolute configurations were investigated by CD spectroscopy. Ceramicine W (3) exhibited potent antimalarial activity against Plasmodium falciparum 3D7 strain with IC 50 value of 1.2 µM. In addition, the structure-antimalarial activity relationship (SAR) of the ceramicines was investigated to identify substituent patterns that may enhance activity. It appears that ring B and the functional groups in the vicinity of rings B and C are critical for the antimalarial activity of the ceramicines. In particular, bulky ester substituents with equatorial orientation at C-7 and C-12 greatly increase the antimalarial activity., (© 2023. The Author(s) under exclusive licence to The Japanese Society of Pharmacognosy.)

Ceramicines are a series of limonoids that were isolated from the bark of Malaysian Chisocheton ceramicus (Meliaceae) and were known to show various biological activity. Four new limonoids, ceramicines Q-T (1-4) were isolated from the barks of C. ceramicus, and their structures were determined on the basis of the 1D and 2D NMR analyses in combination with calculated 13 C chemical shift data. Ceramicines Q-T (1-4) were established to be new limonoids with a cyclopentanone[α]phenanthren ring system with a β-furyl ring at C-17, and without a tetrahydrofuran ring like ceramicine B, which is characteristic of known ceramicines. Ceramicine R (2) exhibited potent antimalarial activity against Plasmodium falciparum 3D7 strain with IC 50 value of 2.8 µM., (© 2023. The Author(s) under exclusive licence to The Japanese Society of Pharmacognosy.)

Rapid diagnostic tests (RDTs) based on immunochromatographic detection of Plasmodium falciparum histidine-rich protein 2 (HRP2) have been frequently used for malaria diagnosis. The HRP2-based RDTs are highly sensitive and easy to use; however, their sensitivity may be low in detecting P. falciparum strains carrying deletion of the pfhrp2 and pfhrp3 genes encoding HRP2 and HRP3, respectively. The automated hematology analyzer XN-31, developed by Sysmex (Kobe, Japan) to aid in malaria diagnosis, has higher sensitivity than RDTs owing to a unique automated nucleic acid staining technology that has shown great potential in clinical settings. In this study, we compared the performance of the XN-31 analyzer and two RDTs to detect pfhrp2- and/or pfhrp3-deleted parasites cultured in vitro. The analyses showed that the analyzer was not only as sensitive to pfhrp2- and/or pfhrp3-deleted strains as it was to the wild-type strain but also had higher sensitivity than the RDTs. These results suggested that the XN-31 analyzer is useful for rapid and reliable detection of pfhrp2- and/or pfhrp3-deleted parasites in clinical settings., Competing Interests: Declaration of Competing Interest F.H. is an employee of Sysmex Corporation., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Bioactivity guided separation of Chukrasia velutina root methanolic extract led to the isolation of nine new isopimarane diterpenoids, chukranoids A-I (1-9). The absolute configuration was then assigned by comparing the experimental CD spectra and the calculated CD spectra. Chukranoids A-I (1-9) showed moderate antimalarial activity against Plasmodium falciparum 3D7 strain. It seems that conjugate system in the isopimarane skeleton may influence their antimalarial activity., (© 2022. The Author(s) under exclusive licence to The Japanese Society of Pharmacognosy.)

Background: A blood-stage vaccine targeting the erythrocytic-stages of the malaria parasite Plasmodium falciparum could play a role to protect against clinical disease. Antibodies against the P. falciparum serine repeat antigen 5 (SE47 and SE36 domains) correlate well with the absence of clinical symptoms in sero-epidemiological studies. A previous phase Ib trial of the recombinant SE36 antigen formulated with aluminum hydroxyl gel (BK-SE36) was promising. This is the first time the vaccine candidate was evaluated in young children below 5 years using two vaccination routes., Methods: Safety and immunogenicity of BK-SE36 was assessed in a double-blind, randomized, controlled, age de-escalating phase Ib trial. Fifty-four Burkinabe children in each age cohort, 25-60 or 12-24 months, were randomized in a 1:1:1 ratio to receive three doses of BK-SE36 either by intramuscular (BK IM) or subcutaneous (BK SC) route on Day 0, Week 4, and 26; or the control vaccine, Synflorix ® via IM route on Day 0, Week 26 (and physiological saline on Week 4). Safety data and samples for immunogenicity analyses were collected at various time-points., Results: Of 108 subjects, 104 subjects (96.3%) (Cohort 1: 94.4%; Cohort 2: 98.1%) received all three scheduled vaccine doses. Local reactions, mostly mild or of moderate severity, occurred in 99 subjects (91.7%). The proportion of subjects that received three doses without experiencing Grade 3 adverse events was similar across BK-SE36 vaccines and control arms (Cohort 1: 100%, 89%, and 89%; and Cohort 2: 83%, 82%, and 83% for BK IM, BK SC, and control, respectively). BK-SE36 vaccine was immunogenic, inducing more than 2-fold change in antibody titers from pre-vaccination, with no difference between the two vaccination routes. Titers waned before the third dose but in both cohorts titers were boosted 6 months after the first vaccination. The younger cohort had 2-fold and 4-fold higher geometric mean titers compared to the 25- to 60-month-old cohort after 2 and 3 doses of BK-SE36, respectively., Conclusion: BK-SE36 was well tolerated and immunogenic using either intramuscular or subcutaneous routes, with higher immune response in the younger cohort., Clinical Trial Registration: https://pactr.samrc.ac.za/TrialDisplay.aspx?TrialID=934, identifier PACTR201411000934120., Competing Interests: TH is the inventor of BK-SE36 and all rights have now been turned over to NPC. NP served as contract researcher for NPC, Apr - Sept 2017. SH, FD and OL received support from NPC for salaries, travel and CRO cost for clinical monitoring. EB, SaC, AD, AZO, JY also received support from NPC for salaries during the long term follow-up. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bougouma, Palacpac, Tiono, Nebie, Ouédraogo, Houard, Yagi, Coulibaly, Diarra, Tougan, Ouedraogo, Soulama, Arisue, Yaro, D’Alessio, Leroy, Cousens, Horii and Sirima.)

Bioactivity-guided separation of the methanol extract of Calophyllum scriblitifolium bark led to the isolation of five new pyranocoumarins, caloforines A-E (1-5) and two new coumarins, caloforines F and G (6 and 7). Their structures were elucidated by 1D and 2D NMR spectroscopy, and their absolute configurations were investigated by a combination of CD spectroscopy and DFT calculation. Caloforines A-F (1-6) showed moderate antimalarial activity against Plasmodium falciparum 3D7 strain., (© 2022. The Author(s) under exclusive licence to The Japanese Society of Pharmacognosy.)

Background: The postoperative imaging assessment of Cochlear Implant (CI) patients is imperative. The main obstacle is that Magnetic Resonance imaging (MR) is contraindicated or hindered by significant artefacts in most cases with CIs. This study describes an automatic cochlear image registration and fusion method that aims to help radiologists and surgeons to process pre-and postoperative 3D multimodal imaging studies in cochlear implant (CI) patients., Methods and Findings: We propose a new registration method, Automatic Cochlea Image Registration (ACIR-v3), which uses a stochastic quasi-Newton optimiser with a mutual information metric to find 3D rigid transform parameters for registration of preoperative and postoperative CI imaging. The method was tested against a clinical cochlear imaging dataset that contains 131 multimodal 3D imaging studies of 41 CI patients with preoperative and postoperative images. The preoperative images were MR, Multidetector Computed Tomography (MDCT) or Cone Beam Computed Tomography (CBCT) while the postoperative were CBCT. The average root mean squared error of ACIR-v3 method was 0.41 mm with a standard deviation of 0.39 mm. The results were evaluated quantitatively using the mean squared error of two 3D landmarks located manually by two neuroradiology experts in each image and compared to other previously known registration methods, e.g. Fast Preconditioner Stochastic Gradient Descent, in terms of accuracy and speed., Conclusions: Our method, ACIR-v3, produces high resolution images in the postoperative stage and allows for visualisation of the accurate anatomical details of the MRI with the absence of significant metallic artefacts. The method is implemented as an open-source plugin for 3D Slicer tool., Competing Interests: The authors have declared that no competing interests exist.

Eight new limonoids, walsogynes H-O (1-8) were isolated from the barks of Walsura chrysogyne, and their structures were determined on the basis of the 1D and 2D NMR data. Walsogynes H-M (1-6) and O (8) were concluded to be 11,12-seco limonoids with a dodecahydro-1H-naphtho[1,8-bc:3,4-c']difuran skeleton, and walsogyne N (7) to be 11,12-seco limonoid sharing a unique dodecahydronaphtho[1,8-bc:5,4-b'c']difuran skeleton. Walsogynes H-O (1-8) exhibited potent antimalarial activity against Plasmodium falciparum 3D7 strain with IC 50 value of 2.5, 2.6, 1.6, 2.5, 1.5, 2.6, 2.1, and 1.1 µM, respectively., (© 2021. The Japanese Society of Pharmacognosy.)

Two new bisindole alkaloids, bisnaecarpamines A (1) and B (2), possessing a vobasine-sarpagine type skeleton were isolated from the bark of Tabernaemontana macrocarpa Jack. Their structures were elucidated by extensive spectroscopic methods and chemical correlation. The absolute configurations of compounds 1 and 2 were established using TDDFT-ECD calculation of the selected isomers. Bisnaecarpamine A exhibited potent antimalarial activity against Plasmodium falciparum 3D7 strain with IC 50 value of 28.8 µM.

Two new bisindole alkaloids, 12'-O-demethyl-vobtusine-5-lactam and isovobtusine-N-oxide (1 and 2), were isolated from the leaves of Voacanga grandifolia, together with two known bisindole alkaloids. Their structures were elucidated on the basis of 1D and 2D NMR data. 1 and 2 showed potent antimalarial activity against Plasmodium falciparum 3D7 and very low cytotoxic activity against a human cell line, HepG2 cells.

The automated hematology analyzers XN-30 (for research) and XN-31 prototype (for diagnosis support) can easily and rapidly detect Plasmodium-infected red blood cells (iRBCs) and distinguish the developmental stages of the parasite in approximately 1 min. Two dedicated reagents, Lysercell M and Fluorocell M, are available with the analyzers. Lysercell M plays an indispensable role in enhancing the fluorescence intensity of the nucleic acid staining dye in Fluorocell M and altering cell morphology. These effects of Lysercell M have been empirically determined but insufficiently analyzed. In this study, the properties of Lysercell M were analyzed using two flow cytometers and a fluorescence microscope. First, the fluorescence intensity emitted by iRBCs treated with Lysercell M or phosphate-buffered saline (PBS) was evaluated. Second, the size of RBCs treated with Lysercell M or PBS was measured. Finally, the morphology of individual parasites was observed after reconstruction of an M scattergram, a cytogram of the XN-31 prototype system, using an imaging flow cytometer. These analyses showed that treatment of iRBCs with Lysercell M increased the fluorescence intensity of stained parasite nucleic acids by approximately 10-fold and reduced the size of iRBCs in a stage-specific manner, facilitating the identification and quantification of ring form, trophozoite, and schizont stage iRBCs. These properties suggest that Lysercell M is useful for rapidly detecting iRBCs and accurately distinguishing the parasite developmental stages, thereby contributing to the usability of the XN-30 and XN-31 prototype analyzers., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: BK-SE36 is blood-stage malaria vaccine candidate that is undergoing clinical trials. Here, the safety and immunogenicity of BK-SE36 with a novel adjuvant, CpG-ODN(K3) (thus, BK-SE36/CpG) was assessed in a phase 1a trial in Japan., Methods: An investigator-initiated, randomised, single-blind, placebo-controlled, dose-escalation study was conducted at Osaka University Hospital with 26 healthy malaria naïve Japanese male adults. The trial was conducted in two stages: Stage/Group 1, half-dose (n = 7 for BK-SE36/CpG and n = 3 for control) and Stage/Group 2, full-dose (n = 11 for BK-SE36/CpG and n = 5 for control). There were two intramuscular vaccinations 21 days apart for both half-dose (0.5 ml: 50 µg SE36 + 500 µg aluminum + 500 µg K3) and full-dose (1.0 ml: 100 µg SE36 + 1000 µg aluminum + 1000 µg K3). A one-year follow-up was done to monitor changes in autoimmune markers and vaccine-induced antibody response., Results: BK-SE36/CpG was well tolerated. Vaccination site reactions were similar to those observed with BK-SE36. During the trial and follow-up period, no subject had clinical evidence of autoimmune disease. The full-dose group had significantly higher titres than the half-dose group (Student's t-test, p = 0.002) at 21 days post-second vaccination. Antibody titres remained above baseline values during 12 months of follow-up. The vaccine induced antibody was mostly composed of IgG1 and IgM, and recognised epitopes close to the polyserine region located in the middle of SE36., Conclusions: BK-SE36/CpG has an acceptable safety profile. Use of CpG-ODN(K3) greatly enhanced immunogenicity in malaria naïve Japanese adults when compared to BK-SE36 alone. The utility of BK-SE36/CpG is currently under evaluation in a malaria endemic setting in West Africa., Trial Registration: JMACCT Clinical Trial Registry JMA-IIA00109., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [T. Horii, inventor and patent holder of SE36; T. Horii, K.J. Ishii and T. Tougan inventors and patent holders of BK-SE36/CpG]., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Iron deficiency is the most common and widespread nutritional disorder worldwide. The automated haematology analyser XN-30 (Sysmex, Kobe, Japan) was developed to detect malaria-infected red blood cells (RBCs) in human blood samples using flow cytometry. The optical system of the analyser detects autofluorescence (AF)-emitting RBCs containing iron-deficient haem groups and would aid in the diagnosis of anaemia resulting from iron deficiency. Here, an RBC-optical (RBO) channel was devised and implemented on the analyser. In vitro analyses showed that the analyser detected AF-emitting RBCs treated with 5-aminolevulinic acid. Furthermore, the analyser detected AF-emitting RBCs in mice fed a low iron diet and infected with a rodent malaria parasite; it could also be effectively used in humans. This study demonstrates that the analyser can quantitatively and reproducibly detect AF-emitting RBCs and measure other haematological parameters, suggesting its usefulness for the initial evaluation of latent iron deficiency anaemia in conjunction with the diagnosis of malaria.

Background: The malaria parasite Plasmodium falciparum is a protozoan that develops in red blood cells (RBCs) and requires various host factors. For its development in RBCs, nutrients not only from the RBC cytosol but also from the extracellular milieu must be acquired. Although the utilization of host nutrients by P. falciparum has been extensively analysed, only a few studies have reported its utilization of host serum proteins. Hence, the aim of the current study was to comprehensively identify host serum proteins taken up by P. falciparum parasites and to elucidate their role in pathogenesis., Methods: Plasmodium falciparum was cultured with human serum in vitro. Uptake of serum proteins by parasites was comprehensively determined via shotgun liquid chromatography-mass spectrometry/mass spectrometry and western blotting. The calcium ion concentration in serum was also evaluated, and coagulation activity of the parasite lysate was assessed., Results: Three proteins, vitamin K-dependent protein S, prothrombin, and vitronectin, were selectively internalized under sufficient Ca 2+ levels in the culture medium. The uptake of these proteins was initiated before DNA replication, and increased during the trophozoite and schizont stages, irrespective of the assembly/disassembly of actin filaments. Coagulation assay revealed that prothrombin was activated and thereby induced blood coagulation., Conclusions: Serum proteins were taken up by parasites under culture conditions with sufficient Ca 2+ levels. This uptake phenomenon was associated with their pathogenicity.

Serine repeat antigen (SERA) is conserved among species of the genus Plasmodium. Sera genes form a multigene family and are generally tandemly clustered on a single chromosome. Although all Plasmodium species encode multiple sera genes, the number varies between species. Among species, the members share similar sequences and gene organization. SERA possess a central papain-like cysteine protease domain, however, in some members, the active site cysteine residue is substituted with a serine. Recent studies implicate this gene family in a number of aspects in parasite biology and induction of protective immune response. This review summarizes the current understanding on this important gene family in several Plasmodium species. The Plasmodium falciparum (Pf)-sera family, for example, consists of nine gene members. Unlike other multigene families in Plasmodium species, Pf-sera genes do not exhibit antigenic variation. Pf-sera5 nucleotide diversity is also low. Moreover, although Pf-sera5 is highly transcribed during the blood stage of malaria infection, and a large amount is released into the host blood following schizont rupture, in malaria endemic countries the sero-positive rates for Pf-SERA5 are low, likely due to Pf-SERA5 binding of host proteins to avoid immune recognition. As an antigen, the N-terminal 47 kDa domain of Pf-SERA5 is a promising vaccine candidate currently undergoing clinical trials. Pf-SERA5 and Pf-SERA6, as well as P. berghei (Pb)-SERA3, and Pb-SERA5, have been investigated for their roles in parasite egress. Two P. yoelii SERA, which have a serine residue at the protease active center, are implicated in parasite virulence. Overall, these studies provide insight that during the evolution of the Plasmodium parasite, the sera gene family members have increased by gene duplication, and acquired various functions that enable the parasite to survive and successfully maintain infection in the host.

Two new sarpagine-type indole alkaloids (1 and 2), together with five known alkaloids; 12-methoxy-4-methylvoachalotine (3), 16-demethoxycarbonylvoacamine (4), isositsirikine (5), affinisine (6), affinine (7), were isolated from the bark of Tabernaemontana macrocarpa Jack. The structures of these alkaloids were determined based on spectroscopic data, chemical correlation, and comparison with the literature. 16-Demethoxycarbonylvoacamine (4) showed antiplasmodial activities against Plasmodium falciparum 3D7 and cytotoxic activities against human cell line, HepG2 cells.

Background: Basic blue 3 is a promising anti-malarial lead compound based on the π-delocalized lipophilic cation hypothesis. Its derivatives with nitrogen atoms bonded to carbon atoms at the 3- and 7-positions on the phenoxazine ring were previously shown to exert potent antiprotozoal activity against Plasmodium falciparum, Trypanosoma cruzi, Trypanosoma brucei rhodesiense, and Leishmania donovani parasites in vitro. However, compounds with nitrogen modification at the 10-position on the phenoxazine ring were not evaluated., Methods: Six acylphenoxazine derivatives (ITT-001 to 006) with nitrogen modification at the 10-position on the phenoxazine ring, which were synthesized from basic blue 3, were characterized and evaluated for anti-malarial activity in vitro with an automated haematology analyzer (XN-30) and light microscopy. Intensity of self-fluorescence was measured using a fluorometer. Localization of basic blue 3 was observed by fluorescence microscopy. Cytotoxicity was evaluated using human cell lines, HEK293T and HepG2 cells. Finally, anti-malarial activity was evaluated in a rodent malaria model., Results: All the six derivatives showed anti-malarial efficacy even against chloroquine-, pyrimethamine-, and artemisinin-resistant field isolates similar to the sensitive strains and isolates in vitro. The efficacy of basic blue 3 was the strongest, followed by that of ITT-001 to 004 and 006, while that of ITT-005 was the weakest. Basic blue 3 showed strong self-fluorescence, whereas ITT derivatives had five- to tenfold lower intensity than that of basic blue 3, which was shown by fluorescence microscopy to be selectively accumulated in the plasmodial cytoplasm. In contrast, ITT-003, 004, and 006 exhibited the lowest cytotoxicity in HEK293T and HepG2 cells in vitro and the highest selectivity between anti-malarial activity and cytotoxicity. The in vivo anti-malarial assay indicated that oral administration of ITT-004 was the most effective against the rodent malaria parasite, Plasmodium berghei NK65 strain., Conclusions: The six ITT derivatives were effective against chloroquine- and pyrimethamine-resistant strains and artemisinin-resistant field isolates as well as the sensitive ones. Among them, ITT-004, which had high anti-malarial activity and low cytotoxicity in vitro and in vivo, is a promising anti-malarial lead compound.

Two new bisindole alkaloids, leucophyllinines A (1) and B (2) consisting of eburnane and quebrachamine-type skeletons were isolated from the bark of Leuconotis eugeniifolia, and their structures were elucidated on the basis of spectroscopic data. Leucophyllinines A and B showed antiplasmodial activities against Plasmodium falciparum 3D7.

Background: The erythrocytic stage of Plasmodium falciparum parasites in humans is clinically important, as the parasites at this growth stage causes malarial symptoms. Most of the currently available anti-malarial drugs target this stage, but the emergence and spread of parasites resistant to anti-malarial drugs are a major challenge to global eradication efforts; therefore, the development of novel medicines is urgently required. In this study, the in vitro anti-malarial activity of five current anti-malarial drugs (artemisinin, atovaquone, chloroquine, mefloquine, and pyrimethamine) and 400 compounds from the Pathogen Box provided by the Medicines for Malaria Venture on P. falciparum parasites was characterized using the XN-30 analyzer. Furthermore, the outcomes obtained using the analyser were classified according to the parasitaemias of total and each developmental stages., Results: The growth inhibition rate and the half-maximal (50%) inhibitory concentration (IC 50 ) of the five current anti-malarial drugs were calculated from the parasitaemia detected using the XN-30 analyzer. Respective strains and drugs presented strongly fitted sigmoidal curves, and the median SD at all tested concentrations was 1.6, suggesting that the variation in values measured with the analyser was acceptably low for the comparison of drug efficacy. Furthermore, the anti-malarial activity of the 400 compounds from the Pathogen Box was tested, and 141 drugs were found to be effective. In addition, the efficacy was classified into 4 types (Type I, parasites were arrested or killed without DNA replication; Type II, parasites were arrested or killed similar to Type I, and the parasitaemia was apparently decreased; Type III, parasites progressed to trophozoite without sufficient DNA replication; and Type IV, parasites were arrested at late trophozoite or schizont after DNA replication)., Conclusion: The current study demonstrates that the XN-30 analyzer objectively, reproducibly, and easily evaluated and characterized the anti-malarial efficacy of various compounds. The results indicate the potential of the XN-30 analyzer as a powerful tool for drug discovery and development in addition to its use as an important diagnostic tool.

The asexual blood stages of the Plasmodium falciparum parasite are responsible for inducing the clinical symptoms and the most severe presentations of malaria infection that causes frequent mortality and morbidity in tropical and subtropical areas of the world, making the blood stages of infection a key target of new malaria treatment and prevention strategies. Progress towards the development of more effective treatment and prevention strategies has been hindered by the limited availability of infection models that permit the sequential analysis of blood stage parasites in vitro followed by in vivo analysis to confirm therapeutic benefits. To advance a model for in vitro and in vivo analysis of blood stage parasites, we examined nine laboratory strains of P. falciparum to determine which strains could adapt to growth in vivo in splenectomized squirrel monkeys (Saimiri sciureus). Only one of the nine laboratory strains tested, the FCB strain, adapted to in vivo growth. Morphological analysis show that the adapted ring-stage parasites have a different morphology from original parasites cultured in vitro, and more often they were found to localize at the edge of the infected red blood cell. No remarkable differences were observed for both trophozoites and schizonts. The adapted strain can be cultured back in vitro similar to the original parasite, indicating that the adapted parasite can develop both in vitro and in vivo. This squirrel monkey-adapted P. falciparum parasite is expected to be suitable and is advantageous for the research and development of vaccines and antimalarial drugs., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: The erythrocytic stage, where malaria parasites proliferate in human blood, is clinically significant as this causes the symptoms and illness of malaria. Experimental rodent models of malaria at the erythrocytic stage are used for the development of anti-malarial drugs and for biological analysis. An automated haematology analyzer XN-30 was developed for detection of infected red blood cells (iRBCs) in human blood samples and measurement of their parasitaemia in approximately 1 min through flow cytometry analysis. Additionally, the analyzer simultaneously measured other haematological parameters in these samples. It is inferred that the analyzer would also allow easy and rapid measurement of parasitaemia in mice and provide important clues on the mouse haematological state during infection and treatment., Results: The XN-30 analyzer is a simple and rapid tool to detect iRBCs in mouse blood samples infected with rodent malarial parasites, with three-dimensional analysis permitting the precise measurement of parasitaemia (referred herein as the 'XN-30 system'). The XN-30 analyzer allowed not only the detection of iRBCs but also the monitoring of RBC, white blood cell, and platelet counts, as well as haematocrit, mean corpuscular volume and mean platelet volume values in the mouse blood sample. For anti-malarial drug development, aside from demonstrating possible efficacy in mouse models, XN-30 analyzer could provide a first glimpse of the safety profile of the drug., Conclusions: The XN-30 system is a powerful tool that can be utilized for the in vivo screening, development, and evaluation of anti-malarial drugs as well as for pre-clinical pharmacology and/or toxicity tests in rodent models.

The malaria parasite Plasmodium falciparum proliferates in the blood stream where the host immune system is most active. To escape from host immunity, P. falciparum has developed a number of evasion mechanisms. Serine repeat antigen 5 (SERA5) is a blood stage antigen highly expressed at late trophozoite and schizont stages. The P47 N-terminal domain of SERA5, the basis of SE36 antigen of the blood stage vaccine candidate under clinical trials, covers the merozoite surface. Exploring the role of the P47 domain, screening of serum proteins showed that vitronectin (VTN) directly binds to 20 residues in the C-terminal region of SE36. VTN co-localized with P47 domain in the schizont and merozoite stages. Phagocytosis assay using THP-1 cells demonstrated that VTN bound to SE36 prevented engulfment of SE36-beads. In addition, several serum proteins localized on the merozoite surface, suggesting that host proteins camouflage merozoites against host immunity via binding to VTN.

Background: The automated haematology analyzer XN-30 (Sysmex, Kobe, Japan) easily and rapidly detects malarial parasites in clinical blood samples using flow cytometry. The XN-30 analyzer is able to distinguish each developmental stage by measuring DNA content and cell size. Thus, it was expected to be capable of quantifying the developmental stages of cultured falciparum parasite. To achieve this requirement, a modified algorithm was tested for its validity and reliability using in vitro cultured falciparum parasite., Results: The XN-30 analyzer automatically measured each developmental stage as well as total parasitaemia. Comparison of the parasitaemia obtained using the XN-30 analyzer equipped with the modified algorithm with that obtained using microscopy examination of Giemsa-stained smears revealed the greater sensitivity and reproducibility of the former. The XN-30 analyzer also detected free merozoites and purified gametocytes., Conclusions: The XN-30 analyzer allows the precise recognition and enumeration of total and each developmental stages of cultured falciparum parasites, and permits the sensitive and reproducible calculation of parasitaemia. The results indicate the potential of the XN-30 analyzer for basic research on malarial biology, anti-malarial drug discovery, and evaluation of drug efficacy.

Signal peptide peptidase (SPP) is an intramembrane aspartic protease involved in the maturation of the core protein of hepatitis C virus (HCV). The processing of HCV core protein by SPP has been reported to be critical for the propagation and pathogenesis of HCV. Here we examined the inhibitory activity of inhibitors for γ-secretase, another intramembrane cleaving protease, against SPP, and our findings revealed that the dibenzoazepine-type structure in the γ-secretase inhibitors is critical for the inhibition of SPP. The spatial distribution showed that the γ-secretase inhibitor compound YO-01027 with the dibenzoazepine structure exhibits potent inhibiting activity against SPP in vitro and in vivo through the interaction of Val223 in SPP. Treatment with this SPP inhibitor suppressed the maturation of core proteins of all HCV genotypes without the emergence of drug-resistant viruses, in contrast to the treatment with direct-acting antivirals. YO-01027 also efficiently inhibited the propagation of protozoa such as Plasmodium falciparum and Toxoplasma gondii These data suggest that SPP is an ideal target for the development of therapeutics not only against chronic hepatitis C but also against protozoiasis., Competing Interests: The authors declare no conflict of interest.

Plasmodium gonderi is a primate parasite whose natural host is the African Old World monkeys. Here, we report the draft genome sequence for P. gonderi The data are useful not only for understanding the evolution of malaria but also for allowing the comparative genomics of malaria parasites., (Copyright © 2017 Honma et al.)

Peptide-drug discovery using host-defense peptides becomes promising against antibiotic-resistant pathogens and cancer cells. Here, we customized the therapeutic activity of bovine cathelicidin-5 targeting to bacteria, protozoa, and tumor cells. The membrane dependent conformational adaptability and plasticity of cathelicidin-5 is revealed by biophysical analysis and atomistic simulations over 200 μs in thymocytes, leukemia, and E. coli cell-membranes. Our understanding of energy-dependent cathelicidin-5 intrusion in heterogeneous membranes aided in designing novel loss/gain-of-function analogues. In vitro findings identified leucine-zipper to phenylalanine substitution in cathelicidin-5 (1-18) significantly enhance the antimicrobial and anticancer activity with trivial hemolytic activity. Targeted mutants of cathelicidin-5 at kink region and N-terminal truncation revealed loss-of-function. We ensured the existence of a bimodal mechanism of peptide action (membranolytic and non-membranolytic) in vitro. The melanoma mouse model in vivo study further supports the in vitro findings. This is the first structural report on cathelicidin-5 and our findings revealed potent therapeutic application of designed cathelicidin-5 analogues.

SE36 antigen, derived from serine repeat antigen 5 (SERA5) of Plasmodium falciparum, is a promising blood stage malaria vaccine candidate. Designated as BK-SE36, the SE36 antigen was formulated with aluminum hydroxyl gel (AHG) and produced under Good Manufacturing Practice (GMP) constraints. In a Phase Ib clinical trial and follow-up study in Uganda, the risk for malaria symptoms was reduced by 72% compared with the control group. Although promising, the number of responders to the vaccine in 6-20years-olds was approximately 30% with the majority in the younger cohort. This is in contrast to the phase Ia clinical trial where response to the vaccine was 100% in Japanese malaria naive adults. A consideration that can be of importance is the involvement of host genetic factors that may influence the ability to mount an effective immune response to vaccination as well as susceptibility to malaria infection. We, therefore, analyzed allelic polymorphism of human leukocyte antigen (HLA)-DRB1 alleles using sequence-based typing (SBT). In this study, DRB1 alleles did not influence antibody response to BK-SE36 and the vaccinees susceptibility to clinical malaria., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The malaria vaccine candidate antigen, SE36, is based on the N-terminal 47 kDa domain of Plasmodium falciparum serine repeat antigen 5 (SERA5). In epidemiological studies, we have previously shown the inhibitory effects of SE36 specific antibodies on in vitro parasite growth and the negative correlation between antibody level and malaria symptoms. A phase 1 b trial of the BK-SE36 vaccine in Uganda elicited 72% protective efficacy against symptomatic malaria in children aged 6-20 years during the follow-up period 130-365 days post-second vaccination. Here, we performed epitope mapping with synthetic peptides covering the whole sequence of SE36 to identify and map dominant epitopes in Ugandan adult serum presumed to have clinical immunity to P. falciparum malaria. High titer sera from the Ugandan adults predominantly reacted with peptides corresponding to two successive N-terminal regions of SERA5 containing octamer repeats and serine rich sequences, regions of SERA5 that were previously reported to have limited polymorphism. Affinity purified antibodies specifically recognizing the octamer repeats and serine rich sequences exhibited a high antibody-dependent cellular inhibition (ADCI) activity that inhibited parasite growth. Furthermore, protein structure predictions and structural analysis of SE36 using spectroscopic methods indicated that N-terminal regions possessing inhibitory epitopes are intrinsically unstructured. Collectively, these results suggest that strict tertiary structure of SE36 epitopes is not required to elicit protective antibodies in naturally immune Ugandan adults.

Endemic Burkitt lymphoma (eBL) is linked to Plasmodium falciparum (Pf) infection geographically, but evidence from individual-level studies is limited. We investigated this issue among 354 childhood eBL cases and 384 age-, sex-, and location-matched controls enrolled in Ghana from 1965 to 1994. Immunoglobulin G1 (IgG1) and immunoglobulin G3 (IgG3) antibodies to antigens diagnostic of recent infection Pf histidine-rich protein-II (HRP-II) and 6NANP, Pf-vaccine candidates SE36 and 42-kDa region of the 3D7 Pf merozoite surface protein-1 (MSP-1), and tetanus toxoid were measured by indirect enzyme-linked immunoassay. Odds ratios (ORs) and 95% confidence intervals (CIs) for association with eBL were estimated using unconditional logistic regression. After adjustments, eBL was positively associated with HRP-IIIgG3 seropositivity (adjusted OR: 1.60; 95% CI 1.08-2.36) and inversely associated with SE36IgG1 seropositivity (adjusted OR: 0.37; 95% CI 0.21-0.64) and with tetanus toxoidIgG3 levels equal or higher than the mean (adjusted OR: 0.46; 95% CI 0.32-0.66). Anti-MSP-1IgG3 and anti-6NANPIgG3 were indeterminate. eBL risk was potentially 21 times higher (95% CI 5.8-74) in HRP-IIIgG3-seropositive and SE36IgG1-seronegative responders compared with HRP-IIIgG3-seronegative and SE36IgG1-seropositive responders. Our results suggest that recent malaria may be associated with risk of eBL but long-term infection may be protective.

Background: Up to now a malaria vaccine remains elusive. The Plasmodium falciparum serine repeat antigen-5 formulated with aluminum hydroxyl gel (BK-SE36) is a blood-stage malaria vaccine candidate that has undergone phase 1a trial in malaria-naive Japanese adults. We have now assessed the safety and immunogenicity of BK-SE36 in a malaria endemic area in Northern Uganda., Methods: We performed a two-stage, randomized, single-blinded, placebo-controlled phase 1b trial (Current Controlled trials ISRCTN71619711). A computer-generated sequence randomized healthy subjects for 2 subcutaneous injections at 21-day intervals in Stage1 (21-40 year-olds) to 1-mL BK-SE36 (BKSE1.0) (n = 36) or saline (n = 20) and in Stage2 (6-20 year-olds) to BKSE1.0 (n = 33), 0.5-mL BK-SE36 (BKSE0.5) (n = 33), or saline (n = 18). Subjects and laboratory personnel were blinded. Safety and antibody responses 21-days post-second vaccination (Day42) were assessed. Post-trial, to compare the risk of malaria episodes 130-365 days post-second vaccination, Stage2 subjects were age-matched to 50 control individuals., Results: Nearly all subjects who received BK-SE36 had induration (Stage1, n = 33, 92%; Stage2, n = 63, 96%) as a local adverse event. No serious adverse event related to BK-SE36 was reported. Pre-existing anti-SE36 antibody titers negatively correlated with vaccination-induced antibody response. At Day42, change in antibody titers was significant for seronegative adults (1.95-fold higher than baseline [95% CI, 1.56-2.43], p = 0.004) and 6-10 year-olds (5.71-fold [95% CI, 2.38-13.72], p = 0.002) vaccinated with BKSE1.0. Immunogenicity response to BKSE0.5 was low and not significant (1.55-fold [95% CI, 1.24-1.94], p = 0.75). In the ancillary analysis, cumulative incidence of first malaria episodes with ≥5000 parasites/µL was 7 cases/33 subjects in BKSE1.0 and 10 cases/33 subjects in BKSE0.5 vs. 29 cases/66 subjects in the control group. Risk ratio for BKSE1.0 was 0.48 (95% CI, 0.24-0.98; p = 0.04)., Conclusion: BK-SE36 is safe and immunogenic. The promising potential of BK-SE36, observed in the follow-up study, warrants a double-blind phase 1/2b trial in children under 5 years., Trial Registration: Controlled-Trials.com ISRCTN71619711.