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Background Studies report herpes zoster associated with short-term stroke risk, but it is unclear whether herpes zoster vaccine reduces stroke risk.Methods We performed cohort studies using Clinical Practice Research Datalink Aurum linked to hospital admission, mortality and deprivation data. We included 678 110 adults aged 70–80 years receiving a live attenuated herpes zoster vaccination between 1 September 2013 and 31 December 2019. We conducted three comparisons to a historical unvaccinated cohort, an active comparator and a self-controlled case series (SCCS). The historical cohort comprised 678 110 matched unvaccinated adults between 2007 and 2012. The active comparator comprised 653 373 adults receiving an influenza vaccination between 2013 and 2019 meeting herpes zoster vaccine eligibility. Cox regression was used to estimate HRs for stroke in the subsequent 5 years adjusted for measured confounders, in the vaccinated compared with comparator cohorts. Secondary outcomes included stroke/transient ischaemic attack and myocardial infarction. The SCCS included incident strokes 12 months before or after herpes zoster vaccination to address healthy vaccinee bias.Results We recorded 16 281 and 30 430 incident strokes among vaccinated and unvaccinated historical patients, over a median of 2.7 and 5.0 years follow-up, respectively. The adjusted HR for stroke for herpes zoster vaccination compared with unvaccinated historical patients was 0.84 (95% CI 0.82 to 0.86) and to influenza-vaccinated patients was 0.88 (0.85 to 0.89). However, stroke incidence rates were lower in the herpes zoster-vaccinated group prevaccination compared with both comparator cohorts. The SCCS found no association between herpes zoster vaccination and stroke 30–119 days later.Conclusions Despite our cohort study, with extensive confounder adjustment and an influenza vaccination active comparator, finding herpes zoster vaccination associated with 12%–16% reduced stroke rates, additional analyses suggested this was largely explained by healthy vaccinee bias. Our study illustrates the importance of robust sensitivity analyses and testing the suitability of causal inference tools for observational vaccine studies.

Spontaneous adverse events reporting systems are used internationally to flag new or unexpected adverse drug reactions (ADRs). Disproportionality analysis is a recognised technique, but false alarms may arise. We aimed to determine whether these new ADR signals had subsequently been followed-up with detailed hypothesis-testing studies. We searched PubMed to identify published studies (years 2017–2021) where the authors reported findings of new ADR signals from disproportionality analyses. We used PubMed and forward citation tracking (Google Scholar) to identify any subsequent confirmatory studies of these ADR signals. We screened 414 titles and abstracts and checked the full-text articles of 57 studies. We found signals for 56 suspected new ADRs from 24 drugs. Google Scholar showed that the ADR studies had been cited a median of seven times (range 0–61). However, none of the suspected new ADRs had undergone detailed evaluation in the citing literature. Similarly, our PubMed search did not find any confirmation studies for the 56 suspected new ADRs. Although many suspected new ADR signals have been identified through disproportionality analysis, most signals have not been further verified as being either genuine ADRs or false alarms. Researchers must focus on follow-up studies for these new signals.

Cerebral paragonimiasis (CP), caused by aberrant migration of Paragonimus worms, frequently invokes serious illness. The causal relationship between the lesion characteristics and patients' symptoms has poorly been understood. CP serodiagnosis has not been properly evaluated to date. A total of 111 CP cases were diagnosed in our laboratory between 1982 and 2003. This study retrospectively assessed the clinical and imaging characteristics of the 105 patients along with the evaluation of diagnostic potentials of recombinant P. westermani yolk ferritin (rPwYF) by enzyme-linked immunosorbent assay (ELISA) employing patients' sera and cerebrospinal fluids (CSFs). We analyzed 60 male and 45 female patients; 50 early-stage patients with non-calcified enhancing nodule(s) (median age, 38 years; interquartile range [IQR], 24.75-52; median symptom duration, 0.75 years; IQR, 0.2-2) and 54 chronic cases having calcified lesion(s) (median age, 33 years; IQR, 25-41; median symptom duration, 10 years; IQR, 5-20). One patient showed a normal neuroimage. The patients were largely diagnosed in their 30s. The parietal lobe was most commonly affected, followed by occipital, frontal, and temporal lobes. Twenty-six patients had lesions encompassing ≥ two lobes. The patients complained mainly of seizures, headaches, hemiparesis, and focal neurologic deficits (P < 0.001). Seizures and visual defects were predominant in patients with calcified lesion(s) (P < 0.001). The diagnostic sensitivity and specificity of rPwYF against serum/CSF were 100%/97% and 97.2%/92.5%, respectively. The specific IgG antibody levels against rPwYF in sera and CSFs showed a positive correlation (r = 0.59). The clinical manifestations of the early-stage patients might be associated with cortical lesions or meningeal irritation, while those in the chronic stage were caused by conglomerated space-occupying lesions. rPwYF would be useful for the serodiagnosis of both early and chronic CP cases.

Pathogenic helminths have evolved mechanisms to preserve reproductive function while surviving long-term in the host via robust protective responses. A protective role of antioxidant enzymes in preventing DNA degradation has long been proposed, but little evidence has been provided. Here, we show that omega-class glutathione transferases (GSTOs) are critical for maintaining viability by protecting the reproductive cell DNA of the carcinogenic liver fluke, Clonorchis sinensis. Clonorchis sinensis GSTO (CsGSTO) activities modified by changes in the GSH/GSSG and NADPH/NADP+ molar ratios suppressed the overproduction of reactive oxygen species. CsGSTO1 and CsGSTO2 catalyzed deglutathionylation under physiologic and low-stress conditions (GSH/GSSG ratio of 6:1 or higher) but promoted glutathionylation under high-stress conditions (GSH/GSSG ratio of 3:1 or lower). Gliotoxin-induced functional disruption of CsGSTOs in living C. sinensis reduced the GSH/GSSG molar ratio and increased the production of protein glutathionylation (PSSG) under physiologic and low-stress conditions, indicating that suppression of GSTO function did not affect deglutathionylation. However, the perturbation of CsGSTOs decreased the GSH/GSSG ratio but also reduced PSSG production under high oxidative stress, demonstrating that glutathionylation was impeded. In response to oxidative stimuli, C. sinensis decreased GSTO-specific dehydroascorbate reductase and thiol transferase activities and the GSH/GSSG ratio, while it increased the NADPH/NADP+ ratio and PSSG. CsGSTOs utilized GSH to regulate GSH/GSSG and NADPH/NADP+ recycling and triggered a redox signal leading to nuclear translocation. Nuclear-imported CsGSTOs were modified by glutathionylation to prevent DNA damage. Antibodies specific to CsGSTOs dose-dependently inhibited this process. Disruption of CsGSTOs or the depletion of GSH caused glutathionylation defects, leading to DNA degradation. Our results demonstrate that CsGSTOs and the GSH system play a previously unappreciated role in protecting DNA from oxidative stress.

Abstract Background Echinococcoses, caused by metacestodes of Echinococcus granulosus (cystic echinococcosis; CE) and E. multilocularis (alveolar echinococcosis; AE), represent major emerging parasitic diseases. These enzootic helminthiases invoke significant public health concerns and social burdens in endemic areas. The diseases are prevalent in the Qinghai-Tibetan Plateau, China, while community-based epidemiological studies have been scarcely reported. We surveyed echinococcosis patients in the southeastern Qinghai Province, China, to better understand the concurrent epidemiological situation in this area. Methods During July and August of 2013 and 2014, we screened echinococcosis patients at Yushu and Golog Prefectures, Qinghai Province, China, in a diagnostic campaign. A total of 2856 people (male:female ratio, 1:1.12; mean age, 34.6 years; age range, 6–88 years) were ultrasonographically examined for the presence of hepatic echinococcal cysts. We also collected serum samples from patients and analyzed antibody reactivity against recombinant forms of diverse E. granulosus antigen Bs (rEgAgB1-5) by enzyme-linked immunosorbent assay. Results We detected 134 patients whose imaging scans were compatible with CE (115 cases) and AE (20 patients). One patient might have been infected with both CE and AE. The overall incidence was 4.7% (CE, 4.0%; AE, 0.7%). A large proportion (67.5%) of CE patients was diagnosed at active and transitional CE1-CE3 stages in their late 30s. The AE cases were generally detected at advanced stage in patients at early 20s (60%). Analysis of the receiver operating characteristic curve and Youden’s index indicated that rEgAgB2 was the most promising biomarker, followed by rEgAgB3 and rEgAgB1. Overall, sensitivity and specificity of rEgAgB1-3 were 84.5–92.7% and 91.9–94.6%, respectively. rEgAgB4 and 5 showed low sensitivity with high cross-reactivity. Conclusions Our results strongly suggest that disability-adjusted life years related to echinococcoses in Qinghai-Tibetan areas might be more serious than previously considered. Control and prevention strategy against CE and AE are highly required in these areas. In addition to ultrasonography, serological tests might provide supportive data. However, serological data should be carefully interpreted for differential diagnosis, especially in areas where both CE and AE are co-endemic.

The small liver fluke Clonorchis sinensis causes hepatobiliary ductal infections in humans. Clonorchiasis is characterized histopathologically by ductal dysplasia, hyperplasia and metaplasia, which closely resembles cholangiocarcinoma (CCA). The disruption of programmed cell death is critical for malignant transformation, while molecular events underlying these phenomena have poorly been understood in clonorchiasis-related CCA tumorigenesis. We incorporated recombinant C. sinensis omega-class glutathione transferase (rCsGSTo) 1 or 2 into human intrahepatic biliary epithelial cells (HIBECs) and analyzed pathophysiological alterations of HIBECs upon the application of oxidative stress. rCsGSTos partially but significantly rescued HIBECs from cell death by inhibiting oxidative stress-induced apoptosis (p < 0.01). rCsGSTos modulated transcriptional levels of numerous genes. We analyzed 13 genes involved in programmed cell death (the upregulation of five antiapoptotic and two apoptotic genes, and the downregulation of one antiapoptotic and five apoptotic genes) and 11 genes associated with cell differentiation (the increase in seven and decrease in four genes) that showed significant modifications (p < 0.05). The induction profiles of the mRNA and proteins of these differentially regulated genes correlated well with each other, and mostly favored apoptotic suppression and/or cell differentiation. We detected increased active, phosphorylated forms of Src, PI3K/Akt, NF-κB p65, MKK3/6 and p38 MAPK, but not JNK and ERK1/2. CsGSTos were localized in the C. sinensis-infected rat cholangiocytes, where cytokeratin 19 was distributed. Our results demonstrated that CsGSTos excreted to the biliary lumen are internalized and accumulated in the host cholangiocytes. When cholangiocytes underwent oxidative stressful condition, CsGSTos appeared to be critically involved in both antiapoptotic process and the differentiation of host cholangiocytes through the regulation of target genes following the activation of responsible signal molecules.

Abstract Background Neurocysticercosis (NC) caused by Taenia solium metacestode (TsM) is a serious neurological disease of global concern. Diverse bioactive molecules involved in the long-term survival of TsM might contribute to disease progression. Fasciclin (Fas) is an extracellular protein that mediates adhesion, migration and differentiation of cells by interacting with other molecules. We hypothesized that TsMFas might bind to calcareous corpuscle (CC) through its adhesive property and participate in crucial protein-protein interactions, thus contributing to the creation of a symbiotic interactome network. Methods Two paralogous TsMFas (TsMFas1 and TsMFas2) were isolated, and their molecular properties were characterized. The co-localization pattern of TsMFas1 and TsMFas2 with CC was determined. CC-TsMFas binary complex was generated by incubating CC with recombinant proteins (rTsMFas1 and 2). In vitro binding assay of CC-rTsMFas1 or CC-rTsMFas2 binary complex with TsM cellular proteins extracted from scolex and neck was conducted. Their binding partners were identified through proteomic analysis. Integrated protein-protein interaction networks were established. Results TsMFas1 (6072 bp long) was composed of 15 exons (841 amino acid polypeptide) interrupted by 14 introns. TsMFas2 (5201 bp long) comprised of 11 exons (597 amino acids) and 10 intervening introns. These proteins displayed 22% amino acid sequence identity to each other, but tightly conserved Fas-related domains. Several isoforms of Fas1 and Fas2 proteins might have been expressed through post-translational modifications. They showed adhesion activity with other cells. TsMFas proteins were largely distributed in parenchymal regions of the scolex and bladder wall. These molecules were co-localized with CC, a unique organelle found in platyhelminths. Subsequent proteome analysis of CC-Fas binary complex mediated protein-protein interactions revealed seven protein ligands in the TsM cellular proteins. Their functions were mainly segregated into carbohydrate metabolism (enolase, phosphoenolpyruvate carboxykinase, phosphoglycerate kinase and glyceraldehyde 3-phosphate dehydrogenase) and cytoskeleton/cellular motility (actin, paramyosin and innexin nuc-9). Those proteins had direct (physical) and/or indirect (functional) relationships along with their biochemical properties and biological roles. Conclusion Protein repertoires strongly suggest that TsMFas and CC may symbiotically mediate protein-protein interactions during biological processes to maintain efficacious homeostatic functions and ensure the prolonged survival of TsM in the host.

Cysticercosis, a parasitic disease caused by Taenia solium metacestode (TsM), has a major global public health impact in terms of disability-adjusted life years. The parasite preferentially infects subcutaneous tissue, but may invade the central nervous system, resulting in neurocysticercosis (NC). NC is an important neglected tropical disease and an emerging disease in industrialized countries due to immigration from endemic areas. The prevalence of taeniasis in Korea declined from 0.3%–12.7% during the 1970s to below 0.02% since the 2000s. A survey conducted from 1993 to 2006 revealed that the percentage of tested samples with high levels of specific anti-TsM antibody declined from 8.3% to 2.2%, suggesting the continuing occurrence of NC in Korea. Modern imaging modalities have substantially improved the diagnostic accuracy of NC, and recent advances in the molecular biochemical characterization of the TsM cyst fluid proteome also significantly strengthened NC serodiagnosis. Two glycoproteins of 150 and 120 kDa that induce strong antibody responses against sera from patients with active-stage NC have been elucidated. The 150 kDa protein showed hydrophobic-ligand binding activities and might be critically involved in the acquisition of host-derived lipid molecules. Fasciclin and endophilin B1, both of which play roles in the homeostatic functions of TsM, showed fairly high antibody responses against calcified NC cases. NC is now controllable and manageable. Further studies should focus on controlling late-onset intractable seizures and serological diagnosis of NC patients infected with few worms. This article briefly overviews diagnostic approaches and discusses current issues relating to NC serodiagnosis.

Background There is growing evidence that sleep duration and quality may be associated with cardiovascular harm and mortality. Methods and Results We conducted a systematic review, meta‐analysis, and spline analysis of prospective cohort studies that evaluate the association between sleep duration and quality and cardiovascular outcomes. We searched MEDLINE and EMBASE for these studies and extracted data from identified studies. We utilized linear and nonlinear dose‐response meta‐analysis models and used DerSimonian–Laird random‐effects meta‐analysis models of risk ratios, with inverse variance weighting, and the I2 statistic to quantify heterogeneity. Seventy‐four studies including 3 340 684 participants with 242 240 deaths among 2 564 029 participants who reported death events were reviewed. Findings were broadly similar across both linear and nonlinear dose‐response models in 30 studies with >1 000 000 participants, and we report results from the linear model. Self‐reported duration of sleep >8 hours was associated with a moderate increased risk of all‐cause mortality, with risk ratio, 1.14 (1.05–1.25) for 9 hours, risk ratio, 1.30 (1.19–1.42) for 10 hours, and risk ratio, 1.47 (1.33–1.64) for 11 hours. No significant difference was identified for periods of self‐reported sleep

Abstract Background Cystic echinococcosis (CE), caused by Echinococcus granulosus metacestode, invokes a serious public health concern. Early diagnosis has great impacts on reduction of disability-adjusted life years. Several antigen B-related molecules (EgAgB; EgAgB1-5) are known to be immunopotent, but detection of EgAgB is variable in many patients and may not allow reliable interpretation of its immunological relevance. More importantly, the immunoproteome profile of hydatid fluid (HF) has not been addressed. Methods We conducted a proteome analysis of the HF of a single fertile cyst of CE1 and CE2 stages through two-dimensional electrophoresis (2-DE). Each protein spot was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). We subsequently determined the immunoproteome profile employing patient sera of entire disease spectrum from CE1 to CE5 stages. Results We identified 40 parasite proteins, of which EgAgB (28 spots) and antigen 5 (EgAg5; 5 molecules) were abundant. EgAgB proteoforms constituted the majority, mostly EgAgB1 (24 spots), followed by EgAgB2 and EgAgB4 (2 spots each). EgAgB3 was detected only by liquid chromatography-MS/MS. EgAgB5 was not recognized. We also detected 38 host proteins, which were largely composed of serum components, antioxidant/xenobiotic enzymes, and enzymes involved in carbohydrate metabolism. CE1 and CE2 HF exhibited comparable spotting patterns, but CE2 HF harbored greater amounts of EgAgB and EgAg5 complexes. CE sera demonstrated complicated immune recognition patterns according to the disease progression; CE2 and CE3 stages exhibited strong antibody responses against diverse EgAgB and EgAg5 proteoforms, while CE1, CE4, and CE5 stages mainly reacted to EgAg5 and cathepsin B. Patient sera of alveolar echinococcosis (AE) cross-reacted with diverse EgAgB isoforms (36%). EgAg5 and cathepsin B also demonstrated cross-reactions with sera from neurocysticercosis and sparganosis. Conclusions Our results demonstrated that detection of a single defined molecule may not properly diagnose CE, since specific immunodominant epitopes changed as the disease progresses. Immunoproteome analysis combined with imaging studies may be practical in the differential diagnosis of CE from AE and other cystic lesions, as well as for staging CE, which are pertinent to establish appropriate patient management.

Background: Parasite proteases have important roles in cleavage of host proteins during the invasion of host tissues and participate in the parasite’s evasion from the host’s immune response. The aim of the present study was to estimate a metalloproteinase properties of Taenia solium metacestode (TsMP) during host-parasite interactions, and evaluate its potential as a serodiagnostic antigen for cysticercosis. Methods: The cDNA coding for the mature catalytic domain of TsMP was cloned into pGEX-6P-1 expression vector. A recombinant glutathione S-transferase and TsMP fusion protein was induced. After refolding and purification, enzymatic properties of the recombinant metalloproteinase were observed. Immunoblot assay was processed to evaluate its potential as a serodiagnostic antigen for cysticercosis. Results: The recombinant TsMP protein showed proteolytic activity, which preferred host extracellular matrix proteins such as collagen and fibronectin as degradable substrates. In immunoblot assay, 87.5% of sera from patients with cysticercosis showed strong reactivity. In sera from patients with other parasitic infections and from normal controls, it showed high specificity. Conclusions: TsMP might be involved in the processing of numerous host proteins and play an important role in the parasite life cycle. A single recombinant TsMP antigen could have a potential value for serodiagnosis of cysticercosis.

Long-term inhaled corticosteroids (ICS) may reduce growth velocity and final height of children with asthma. We aimed to evaluate the association between ICS use of >12 months and growth.We initially searched MEDLINE and EMBASE in July 2013, followed by a PubMed search updated to December 2014. We selected RCTs and controlled observational studies of ICS use in patients with asthma. We conducted random effects meta-analysis of mean differences in growth velocity (cm/year) or final height (cm) between groups. Heterogeneity was assessed using the I2 statistic.We found 23 relevant studies (twenty RCTs and three observational studies) after screening 1882 hits. Meta-analysis of 16 RCTs showed that ICS use significantly reduced growth velocity at one year follow-up (mean difference -0.48 cm/year (95% CI -0.66 to -0.29)). There was evidence of a dose-response effect in three RCTs. Final adult height showed a mean reduction of -1.20 cm (95% CI -1.90 cm to -0.50 cm) with budesonide versus placebo in a high quality RCT. Meta-analysis of two lower quality observational studies revealed uncertainty in the association between ICS use and final adult height, pooled mean difference -0.85 cm (95% CI -3.35 to 1.65).Use of ICS for >12 months in children with asthma has a limited impact on annual growth velocity. In ICS users, there is a slight reduction of about a centimeter in final adult height, which when interpreted in the context of average adult height in England (175 cm for men and 161 cm for women), represents a 0.7% reduction compared to non-ICS users.

BACKGROUND: Clonorchis sinensis causes chronic cumulative infections in the human hepatobiliary tract and is intimately associated with cholangiocarcinoma. Approximately 35 million people are infected and 600 million people are at risk of infections worldwide. C. sinensis excretory-secretory products (ESP) constitute the first-line effector system affecting the host-parasite interrelationship by interacting with bile fluids and ductal epithelium. However, the secretory behavior of C. sinensis in an environment close to natural host conditions is unclear. C. sinensis differs from Fasciola hepatica in migration to, and maturation in, the hepatic bile duct, implying that protein profile of the ESP of these two trematodes might be different from each other. METHODOLOGY/PRINCIPAL FINDINGS: We conducted systemic approaches to analyze the C. sinensis ESP proteome and the biological reactivity of C. sinensis glutathione transferases (GSTs), such as global expression patterns and induction profiles under oxidative stress and host bile. When we observed ex host excretion behavior of C. sinensis in the presence of 10% host bile, the global proteome pattern was not significantly altered, but the amount of secretory proteins was increased by approximately 3.5-fold. Bioactive molecules secreted by C. sinensis revealed universal/unique features in relation to its intraluminal hydrophobic residing niche. A total of 38 protein spots identified abundantly included enzymes involved in glucose metabolism (11 spots, 28.9%) and diverse-classes of glutathione transferases (GSTs; 10 spots, 26.3%). Cathepsin L/F (four spots, 10.5%) and transporter molecules (three spots, 7.9%) were also recognized. The universal secretory proteins found in other parasites, such as several enzymes involved in glucose metabolism and oxygen transporters, were commonly detected. C. sinensis secreted less cysteine proteases and fatty acid binding proteins compared to other tissue-invading or intravascular trematodes. Interestingly, secretion of a 28 kDa σ-class GST (Cs28σGST3) was significantly affected by the host bile, involving reduced secretion of the 28 kDa species and augmented secretion of Cs28σGST3-related high-molecular-weight 85 kDa protein. Oxidative stressors induced upregulated secretion of 28 kDa Cs28σGST3, but not an 85 kDa species. A secretory 26 kDa μ-class GST (Cs26μGST2) was increased upon treatment with oxidative stressors and bile juice, while another 28 kDa σ-class GST (Cs28σGST1) showed negligible responses. CONCLUSIONS/SIGNIFICANCE: Our results represent the first analysis of the genuine nature of the C. sinensis ESP proteome in the presence of host bile mimicking the natural host environments. The behavioral patterns of migration and maturation of C. sinensis in the bile ducts might contribute to the secretion of copious amounts of diverse GSTs, but a smaller quantity and fewer kinds of cysteine proteases. The Cs28σGST1 and its paralog(s) detoxify endogenous oxidative molecules, while Cs28σGST3 and Cs26μGST2 conjugate xenobiotics/hydrophobic substances in the extracellular environments, which imply that diverse C. sinensis GSTs might have evolved for each of the multiple specialized functions.

BACKGROUND: Fatty acid (FA) binding proteins (FABPs) of helminths are implicated in acquisition and utilization of host-derived hydrophobic substances, as well as in signaling and cellular interactions. We previously demonstrated that secretory hydrophobic ligand binding proteins (HLBPs) of Taenia solium metacestode (TsM), a causative agent of neurocysticercosis (NC), shuttle FAs in the surrounding host tissues and inwardly transport the FAs across the parasite syncytial membrane. However, the protein molecules responsible for the intracellular trafficking and assimilation of FAs have remained elusive. METHODOLOGY/PRINCIPAL FINDINGS: We isolated two novel TsMFABP genes (TsMFABP1 and TsMFABP2), which encoded 133- and 136-amino acid polypeptides with predicted molecular masses of 14.3 and 14.8 kDa, respectively. They shared 45% sequence identity with each other and 15-95% with other related-members. Homology modeling demonstrated a characteristic β-barrel composed of 10 anti-parallel β-strands and two α-helices. TsMFABP2 harbored two additional loops between β-strands two and three, and β-strands six and seven, respectively. TsMFABP1 was secreted into cyst fluid and surrounding environments, whereas TsMFABP2 was intracellularly confined. Partially purified native proteins migrated to 15 kDa with different isoelectric points of 9.2 (TsMFABP1) and 8.4 (TsMFABP2). Both native and recombinant proteins bound to 11-([5-dimethylaminonaphthalene-1-sulfonyl]amino)undecannoic acid, dansyl-DL-α-amino-caprylic acid, cis-parinaric acid and retinol, which were competitively inhibited by oleic acid. TsMFABP1 exhibited high affinity toward FA analogs. TsMFABPs showed weak binding activity to retinol, but TsMFABP2 showed relatively high affinity. Isolation of two distinct genes from an individual genome strongly suggested their paralogous nature. Abundant expression of TsMFABP1 and TsMFABP2 in the canal region of worm matched well with the histological distributions of lipids and retinol. CONCLUSIONS/SIGNIFICANCE: The divergent biochemical properties, physiological roles and cellular distributions of the TsMFABPs might be one of the critical mechanisms compensating for inadequate de novo FA synthesis. These proteins might exert harmonized or independent roles on lipid assimilation and intracellular signaling. The specialized distribution of retinol in the canal region further implies that cells in this region might differentiate into diverse cell types during metamorphosis into an adult worm. Identification of bioactive systems pertinent to parasitic homeostasis may provide a valuable target for function-related drug design.

Multiple cysteine proteases of malaria parasites are required for maintenance of parasite metabolic homeostasis and egress from the host erythrocyte. In Plasmodium falciparum these proteases appear to mediate the processing of hemoglobin and aspartic proteases (plasmepsins) in the acidic food vacuole and the hydrolysis of erythrocyte structural proteins at neutral pH. Two cysteine proteases, vivapain (VX)-2 and VX-3 have been characterized in P. vivax, but comprehensive studies of P. vivax cysteine proteases remain elusive.We characterized a novel cysteine protease of P. vivax, VX-4, of which orthologs appears to have evolved differentially in primate plasmodia with strong cladistic affinity toward those of rodent Plasmodium. Recombinant VX-4 demonstrated dual substrate specificity depending on the surrounding micro-environmental pH. Its hydrolyzing activity against benzyloxycarbonyl-Leu-Arg-4-methyl-coumaryl-7-amide (Z-Leu-Arg-MCA) and Z-Phe-Arg-MCA was highest at acidic pH (5.5), whereas that against Z-Arg-Arg-MCA was maximal at neutral pH (6.5-7.5). VX-4 preferred positively charged amino acids and Gln at the P1 position, with less strict specificity at P3 and P4. P2 preferences depended on pH (Leu at pH 5.5 and Arg at pH 7.5). Three amino acids that delineate the S2 pocket were substituted in VX-4 compared to VX-2 and VX-3 (Ala90, Gly157 and Glu180). Replacement of Glu180 abolished activity against Z-Arg-Arg-MCA at neutral pH, indicating the importance of this amino acid in the pH-dependent substrate preference. VX-4 was localized in the food vacuoles and cytoplasm of the erythrocytic stage of P. vivax. VX-4 showed maximal activity against actin at neutral pH, and that against P. vivax plasmepsin 4 and hemoglobin was detected at neutral/acidic and acidic pH, respectively.VX-4 demonstrates pH-dependent substrate switching, which might offer an efficient mechanism for the specific cleavage of different substrates in different intracellular environments. VX-4 might function as a hemoglobinase in the acidic parasite food vacuole, a maturase of P. vivax plasmepsin 4 at neutral or acidic pH, and a cytoskeleton-degrading protease in the neutral erythrocyte cytosol.

Spontaneous adverse events reporting systems are used internationally to flag new or unexpected adverse drug reactions (ADRs). Disproportionality analysis is a recognised technique, but false alarms may arise. We aimed to determine whether these new ADR signals had subsequently been followed-up with detailed hypothesis-testing studies. We searched PubMed to identify published studies (years 2017–2021) where the authors reported findings of new ADR signals from disproportionality analyses. We used PubMed and forward citation tracking (Google Scholar) to identify any subsequent confirmatory studies of these ADR signals. We screened 414 titles and abstracts and checked the full-text articles of 57 studies. We found signals for 56 suspected new ADRs from 24 drugs. Google Scholar showed that the ADR studies had been cited a median of seven times (range 0–61). However, none of the suspected new ADRs had undergone detailed evaluation in the citing literature. Similarly, our PubMed search did not find any confirmation studies for the 56 suspected new ADRs. Although many suspected new ADR signals have been identified through disproportionality analysis, most signals have not been further verified as being either genuine ADRs or false alarms. Researchers must focus on follow-up studies for these new signals. [ABSTRACT FROM AUTHOR]