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Background The emergence and spread of Plasmodium falciparum parasites that lack HRP2/3 proteins and the resulting decreased utility of HRP2-based malaria rapid diagnostic tests (RDTs) prompted the World Health Organization and other global health stakeholders to prioritize the discovery of novel diagnostic biomarkers for malaria. Methods To address this pressing need, we adopted a dual, systematic approach by conducting a systematic review of the literature for publications on diagnostic biomarkers for uncomplicated malaria and a systematic in silico analysis of P. falciparum proteomics data for Plasmodium proteins with favorable diagnostic features. Results Our complementary analyses led us to 2 novel malaria diagnostic biomarkers compatible for use in an RDT format: glyceraldehyde 3-phosphate dehydrogenase and dihydrofolate reductase-thymidylate synthase. Conclusions Overall, our results pave the way for the development of next-generation malaria RDTs based on new antigens by identifying 2 lead candidates with favorable diagnostic features and partially de-risked product development prospects., The World Health Organization called for the identification of novel biomarkers that can fill the diagnostic gap in settings where hrp2/3 deletions are common. By adopting a dual approach, we identified 2 candidates, glyceraldehyde 3-phosphate dehydrogenase and dihydrofolate reductase-thymidylate synthase, with favorable diagnostic features.

BackgroundPlasmodium knowlesi causes zoonotic malaria across Southeast Asia. First-line diagnostic microscopy cannot reliably differentiate P. knowlesi from other human malaria species. Rapid diagnostic tests (RDTs) designed for P. falciparum and P. vivax are used routinely in P. knowlesi co-endemic areas despite potential cross-reactivity for species-specific antibody targets.MethodsTen RDTs were evaluated: nine to detect clinical P. knowlesi infections from Malaysia, and nine assessing limit of detection (LoD) for P. knowlesi (PkA1-H.1) and P. falciparum (Pf3D7) cultures. Targets included Plasmodium-genus parasite lactate dehydrogenase (pan-pLDH) and P. vivax (Pv)-pLDH.ResultsSamples were collected prior to antimalarial treatment from 127 patients with microscopy-positive PCR-confirmed P. knowlesi mono-infections. Median parasitaemia was 788/µL (IQR 247-5,565/µL). Pan-pLDH sensitivities ranged from 50.6% (95% CI 39.6–61.5) (SD BIOLINE) to 87.0% (95% CI 75.1–94.6) (First Response® and CareStart™ PAN) compared to reference PCR. Pv-pLDH RDTs detected P. knowlesi with up to 92.0% (95% CI 84.3-96.7%) sensitivity (Biocredit™). For parasite counts ≥200/µL, pan-pLDH (Standard Q) and Pv-pLDH RDTs exceeded 95% sensitivity. Specificity of RDTs against 26 PCR-confirmed negative controls was 100%. Sensitivity of the 6 highest performing RDTs were not significantly different when comparing samples taken before and after (median 3 hours) antimalarial treatment. Parasite ring stages were present in 30% of pre-treatment samples, with ring stage proportions (mean 1.9%) demonstrating inverse correlation with test positivity of Biocredit™ and two CareStart™ RDTs.For cultured P. knowlesi, CareStart™ PAN demonstrated the lowest LoD at 25 parasites/µL; LoDs of other pan-pLDH ranged from 98 to >2000 parasites/µL. Pv-pLDH LoD for P. knowlesi was 49 parasites/µL. P. falciparum-pLDH or histidine-rich-protein-2 channels did not react with P. knowlesi.ConclusionSelected RDTs demonstrate sufficient performance for detection of all human malaria species including P. knowlesi in co-endemic areas where microscopy is not available, particularly for higher parasite counts, although cannot reliably differentiate among non-falciparum malaria.

IntroductionIn 2014, the WHO published high-priority target product profiles (TPPs) for new tuberculosis (TB) diagnostics to align end-user needs with test targets and specifications; nevertheless, no TB test meets these targets to date. The COVID-19-driven momentum in the diagnostics world offers an opportunity to address the long-standing lack of innovation in the field of TB diagnostics. This scoping review aims to summarise point-of-care (POC) molecular and antigen tests for COVID-19 diagnosis that, when applied to TB, potentially meet WHO TPPs. This summary of currently available innovative diagnostic tools will guide the development of novel TB diagnostics toward the WHO-set targets.Methods and analysisWe will follow the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension Scoping Reviews recommendations. MEDLINE (via PubMed), bioRxiv, MedRxiv and other publicly available in vitro diagnostic test databases were searched on 23 November 2022. POC antigen or molecular tests developed for SARS-CoV-2 detection that meet the eligibility criteria will be included in the review. Developer description, test description, operation characteristics, pricing information, performance and commercialisation status of diagnostic tests identified will be extracted using a predefined standardised data extraction form. Two reviewers will independently perform the screening and data extraction. A narrative synthesis of the final data will be provided.Ethics and disseminationNo ethical approval is required because individual patient data will not be included. The findings will be published in open-access scientific journals.

New diagnostic innovations for tuberculosis (TB), including point-of-care solutions, are critical to reach the goals of the End TB Strategy. However, despite decades of research, numerous reports on new biomarker candidates, and significant investment, no well-performing, simple and rapid TB diagnostic test is yet available on the market, and the search for accurate, non-DNA biomarkers remains a priority. To help overcome this 'biomarker pipeline problem', FIND and partners are working on the development of a well-curated and user-friendly TB biomarker database. The web-based database will enable the dynamic tracking of evidence surrounding biomarker candidates in relation to target product profiles (TPPs) for needed TB diagnostics. It will be able to accommodate raw datasets and facilitate the verification of promising biomarker candidates and the identification of novel biomarker combinations. As such, the database will simplify data and knowledge sharing, empower collaboration, help in the coordination of efforts and allocation of resources, streamline the verification and validation of biomarker candidates, and ultimately lead to an accelerated translation into clinically useful tools.

Millions of cases of tuberculosis (TB) go undiagnosed each year. Better diagnostic tools are urgently needed. Biomarker-based or multiple marker biosignature-based tests, ideally performed on blood or urine, for the detection of active TB might help to meet target product profiles proposed by the World Health Organization for point-of-care testing. We conducted a systematic review to summarize evidence on proposed biomarkers and biosignatures and evaluate their quality and level of evidence. We screened the titles and abstracts of 7,631 citations and included 443 publications that fulfilled the inclusion criteria and were published in 2010-2017. The types of biomarkers identified included antibodies, cytokines, metabolic activity markers, mycobacterial antigens and volatile organic compounds. Only 47% of studies reported a culture-based reference standard and diagnostic sensitivity and specificity. Forty-four biomarkers (4%) were identified in high-quality studies and met the target product profile minimum criteria, of which two have been incorporated into commercial assays. Of the 44 highest-quality biomarkers, 24 (55%) were multiple marker biosignatures. No meta-analyses were performed owing to between-study heterogeneity. In conclusion, TB biomarker discovery studies are often poorly designed and findings are rarely confirmed in independent studies. Few markers progress to a further developmental stage. More validation studies that consider the intended diagnostic use cases and apply rigorous design are needed. The extracted data from this review are currently being used by FIND as the foundation of a dynamic database in which biomarker data and developmental status will be presented.

This work summarizes the available data on the performance of rapid diagnostic tests (RDTs) for the detection of monoinfections due to Plasmodium species P. knowlesi, P. malariae, and P. ovale and indicates low performance of RDTs to detect these infections., Background Despite the increased use and worldwide distribution of malaria rapid diagnostic tests (RDTs) that distinguish between Plasmodium falciparum and non-falciparum species, little is known about their performance detecting Plasmodium knowlesi (Pk), Plasmodium malariae (Pm), and Plasmodium ovale (Po). This review seeks to analyze the results of published studies evaluating the diagnostic accuracy of malaria RDTs in detecting Pk, Pm, and Po monoinfections. Methods MEDLINE, EMBASE, Web of Science, and CENTRAL databases were systematically searched to identify studies that reported the performance of RDTs in detecting Pk, Pm, and Po monoinfections. Results Among 40 studies included in the review, 3 reported on Pk, 8 on Pm, 5 on Po, 1 on Pk and Pm, and 23 on Pm and Po infections. In the meta-analysis, estimates of sensitivities of RDTs in detecting Pk infections ranged 2%–48%. Test performances for Pm and Po infections were less accurate and highly heterogeneous, mainly because of the small number of samples tested. Conclusions Limited data available suggest that malaria RDTs show suboptimal performance for detecting Pk, Pm, and Po infections. New improved RDTs and appropriately designed cross-sectional studies to demonstrate the usefulness of RDTs in the detection of neglected Plasmodium species are urgently needed.

In the original version of this Analysis, author Seda Yerlikaya was mistakenly spelled as Seda Yerliyaka. This has now been amended.

Phosphorylation of the S6 protein of the 40S subunit of the eukaryote ribosome downstream of anabolic signals has long been assumed to promote protein synthesis. Both target of rapamycin complexes regulate this modification in yeast, but the use of ribosome profiling shows no role for Rps6 phosphorylation in mRNA translation., Nutrient-sensitive phosphorylation of the S6 protein of the 40S subunit of the eukaryote ribosome is highly conserved. However, despite four decades of research, the functional consequences of this modification remain unknown. Revisiting this enigma in Saccharomyces cerevisiae, we found that the regulation of Rps6 phosphorylation on Ser-232 and Ser-233 is mediated by both TOR complex 1 (TORC1) and TORC2. TORC1 regulates phosphorylation of both sites via the poorly characterized AGC-family kinase Ypk3 and the PP1 phosphatase Glc7, whereas TORC2 regulates phosphorylation of only the N-terminal phosphosite via Ypk1. Cells expressing a nonphosphorylatable variant of Rps6 display a reduced growth rate and a 40S biogenesis defect, but these phenotypes are not observed in cells in which Rps6 kinase activity is compromised. Furthermore, using polysome profiling and ribosome profiling, we failed to uncover a role of Rps6 phosphorylation in either global translation or translation of individual mRNAs. Taking the results together, this work depicts the signaling cascades orchestrating Rps6 phosphorylation in budding yeast, challenges the notion that Rps6 phosphorylation plays a role in translation, and demonstrates that observations made with Rps6 knock-ins must be interpreted cautiously.

PubMedID: 21459434 Immunostimulatory properties of mushroom derived polysaccharides (PS) as stand-alone agents were tested. Next, PS were nanocomplexed with polyI:C (pIC) to yield stable nanoparticles around 200 nm in size evidenced by atomic force microscopy and dynamic light scattering analyses. PSs were selectively engaged by cells expressing TLR2 and initiated NF?B dependent signaling cascade leading to a Th1-biased cytokine/chemokine secretion in addition to bactericidal nitric oxide (NO) production from macrophages. Moreover, cells treated with nanoparticles led to synergistic IL6, production and upregulation of TNF?, MIP3?, IFN? and IP10 transcript expression. In mice, PS-Ovalbumin-pIC formulation surpassed anti-OVA IgG responses when compared to either PS-OVA or pIC-OVA mediated immunity. Our results revealed that signal transduction initiated both by TLR2 and TLR3 via co-delivery of pIC by PS in nanoparticle depot delivery system is an effective immunization strategy. The present work implicate that the PS and nucleic acid based nanoparticle approach along with protein antigens can be harnessed to prevent infectious diseases. © 2011 Elsevier Ltd. 203953 036615 108S316 This work was supported by the Scientific and Technological Research Council of Turkey (TÜBİTAK, Grant No: 108S316 ). IG received grant support from EU/FP6/MC IRG (Grant # 036615 ) and EU 7th Framework Project UNAM-Regpot (Grant # 203953 ). GT and FCY received scholarship grants from SANTEZ ( 0448-STZ-2009-2 ) and TUBITAK . We thank to Dr. Aykutlu Dana and his group members for their invaluable support during AFM studies. Drs. Can K. Akcali and Mayda Gursel are sincerely acknowledged for their critical reading of the manuscript. Appendix

Emerging evidence supports the notion that claudins (Cldns) are dynamically regulated under normal conditions to respond to the selective permeability requirements of various tissues, and that their expression is developmentally controlled. We describe the localization of those Cldns that we have previously demonstrated to be functionally important in epidermal differentiation and the formation of the epidermal permeability barrier, e.g., Cldn1, Cldn6, Cldn11, and Cldn18, and the presence of Cldn3 and Cldn5 in various neonatal mouse epithelia including the epidermis, nail, oral mucosa, tongue, and stomach. Cldn1 is localized in the differentiated and/or undifferentiated compartments of the epidermis and nail and in the dorsal surface of the tongue and glandular compartment of the stomach but is absent from the oral mucosa and the keratinized compartment of the stomach. Cldn3 is present in the basal cells of the nail matrix and both compartments of the murine stomach but not in the epidermis, oral mucosa, or tongue. Cldn5 is found in the glandular compartment of the stomach but not in the epidermis, nail unit, oral mucosa, forestomach, and tongue. Cldn6, Cldn11, and Cldn18 occur in the differentiating suprabasal compartment of the epidermis, nail, and oral mucosa and in the dorsal and ventral surfaces of the tongue and the keratinized squamous epithelium of the stomach. The simple columnar epithelium of the glandular stomach stains for Cldn18 and reveals a non-membranous pattern for Cldn6 and Cldn11 expression. Our results demonstrate differential Cldn protein profiles in various epithelial tissues and their differentiation stages. Although the molecular mechanisms regulating Cldn expression are unknown, elucidation of their differential localization patterns in tissues with diverse permeability requirements should provide a better understanding of the role of tight junctions in tissue function.

Toll-like receptors (TLR) induce NF-κB dependent or independent cytokine/chemokine secretion and contribute to the establishment of innate and adaptive immune response. When nucleic acid ligands were given in vivo they were attacked by endonucleases and have low uptake efficiency into immune cells due to premature adsorption to serum proteins. Polysaccharides (PS) from edible mushrooms were characterized as immunostimulatory amphiphilic macromolecules. Due to their β-glucan moieties, we have established that these PS`s are recognized and induce TLR2 (but not via NOD-1 or NOD2) mediated activation (as evidenced by NO, TNFα IL6 responses) and can be naturally targeted through this extracellular receptor. Next, we complexed PS with pI:C, R848 and CpG ODN, a TLR3,7/8, and 9 ligands. Different ratio of these nanocomplexes displayed a strong synergism as evidenced by a Th1-biased cytokine secretion and NO production compared to their alone counterparts. This study showed that natural PS is an immunostimulatory compound by itself, and furthermore, it could be used to effectively complex and deliver nucleic acid based TLR ligands to relevant immune cells. The targeted delivery of the generated nanocomplexes led to a more pronounced immune activation that is not achievable either of the stimulants when given alone.

TORC1 is a conserved multisubunit kinase complex that regulates many aspects of eukaryotic growth including the biosynthesis of ribosomes. The TOR protein kinase resident in TORC1 is responsive to environmental cues and is potently inhibited by the natural product rapamycin. Recent characterization of the rapamycin-sensitive phosphoproteome in yeast has yielded insights into how TORC1 regulates growth. Here, we show that Sch9, an AGC family kinase and direct substrate of TORC1, promotes ribosome biogenesis (Ribi) and ribosomal protein (RP) gene expression via direct inhibitory phosphorylation of the transcriptional repressors Stb3, Dot6 and Tod6. Deletion of STB3, DOT6 and TOD6 partially bypasses the growth and cell size defects of an sch9 strain and reveals interdependent regulation of both Ribi and RP gene expression, and other aspects of Ribi. Dephosphorylation of Stb3, Dot6 and Tod6 enables recruitment of the RPD3L histone deacetylase complex to repress Ribi/RP gene promoters. Taken together with previous studies, these results suggest that Sch9 is a master regulator of ribosome biogenesis through the control of Ribi, RP, ribosomal RNA and tRNA gene transcription. The EMBO Journal (2011) 30, 3052-3064. doi:10.1038/emboj.2011.221; Published online 5 July 2011