Antibacterial, cytotoxic and trypanocidal activities of marine-derived fungi isolated from Philippine macroalgae and seagrasses

The occurrence and bioactivities of marine-derived fungi are evaluated in this paper. A total of 16 morphospecies of marine-derived fungi (MDF) were isolated from four host macroalgae and two seagrasses and identified as belonging to the genera Aspergillus, Fusarium, Paecilomyces, Penicillium, Sclerotinia, Thamnidium and Trichoderma, including five mycelia sterilia. Among these host organisms, the rhodophyte Laurencia intermedia harboured the highest number of isolated MDF. Selected MDF were then assayed and showed to inhibit Pseudomonas aeruginosa (8-19 mm zone of inhibition) and Staphylococcus aureus (6-19 mm zone of inhibition), and were cytotoxic against the brine shrimp Artemia salina nauplii (LD50: 201.56-948.37 μg mL-1). The screening led to the selection of five of the most bioactive morphospecies, all belonging to the genus Aspergillus. These marine aspergilli were subjected to β-tubulin gene sequence analysis for species identification, and to mass production in different culture media with or without marine salts, and screening of the crude culture extracts for their cytotoxic and trypanocidal activities. Aspergillus tubingensis cultivated in potato dextrose broth with marine salt proved to be the most cytotoxic against P388 (IC50: 1028 ng mL-1) and HeLa (IC50: 1301 ng mL-1) cancer cells. On the other hand, A. fumigatus cultivated in malt extract broth without marine salt was shown to be the most potent against Trypanosoma congolense (IC50: 298.18 ng mL-1). Our study therefore showed that salinity may influence the bioactivities of some species of MDF

Long-COVID Symptoms in Individuals Infected with Different SARS-CoV-2 Variants of Concern:A Systematic Review of the Literature

The association of SARS-CoV-2 variants with long-COVID symptoms is still scarce, but new data are appearing at a fast pace. This systematic review compares the prevalence of long-COVID symptoms according to relevant SARS-CoV-2 variants in COVID-19 survivors. The MEDLINE, CINAHL, PubMed, EMBASE and Web of Science databases, as well as the medRxiv and bioRxiv preprint servers, were searched up to 25 October 2022. Case-control and cohort studies analyzing the presence of post-COVID symptoms appearing after an acute SARS-CoV-2 infection by the Alpha (B.1.1.7), Delta (B.1.617.2) or Omicron (B.1.1.529/BA.1) variants were included. Methodological quality was assessed using the Newcastle–Ottawa Scale. From 430 studies identified, 5 peer-reviewed studies and 1 preprint met the inclusion criteria. The sample included 355 patients infected with the historical variant, 512 infected with the Alpha variant, 41,563 infected with the Delta variant, and 57,616 infected with the Omicron variant. The methodological quality of all studies was high. The prevalence of long-COVID was higher in individuals infected with the historical variant (50%) compared to those infected with the Alpha, Delta or Omicron variants. It seems that the prevalence of long-COVID in individuals infected with the Omicron variant is the smallest, but current data are heterogeneous, and long-term data have, at this stage, an obviously shorter follow-up compared with the earlier variants. Fatigue is the most prevalent long-COVID symptom in all SARS-CoV-2 variants, but pain is likewise prevalent. The available data suggest that the infection with the Omicron variant results in fewer long-COVID symptoms compared to previous variants; however, the small number of studies and the lack of the control of cofounders, e.g., reinfections or vaccine status, in some studies limit the generality of the results. It appears that individuals infected with the historical variant are more likely to develop long-COVID symptomatology

In silico evaluation of the impact of Omicron variant of concern sublineage BA.4 and BA.5 on the sensitivity of RT-qPCR assays for SARS-CoV-2 detection using whole genome sequencing

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant of concern (VoC) Omicron (B.1.1.529) has rapidly spread around the world, presenting a new threat to global public human health. Due to the large number of mutations accumulated by SARS-CoV-2 Omicron, concerns have emerged over potentially reduced diagnostic accuracy of reverse transcription polymerase chain reaction (RT-qPCR), the gold standard diagnostic test for diagnosing coronavirus disease 2019 (COVID-19). Thus, we aimed to assess the impact of the currently endemic Omicron sublineages BA.4 and BA.5 on the integrity and sensitivity of RT-qPCR assays used for coronavirus disease 2019 (COVID-19) diagnosis via in silico analysis. We employed whole genome sequencing data and evaluated the potential for false negatives or test failure due to mismatches between primers/probes and Omicron VoC viral genome. Methods: In silico sensitivity of 12 RT-qPCR tests (containing 30 primers and probe sets) developed for detection of SARS-CoV-2 reported by the World Health Organization (WHO) or available in the literature, was assessed for specifically detecting SARS-CoV-2 Omicron BA.4 and BA.5 sublineages, obtained after removing redundancy from publicly available genomes from National Center for Biotechnology Information (NCBI) and Global Initiative on Sharing Avian Influenza Data (GISAID) databases. Mismatches between amplicon regions of SARS-CoV-2 Omicron VoC and primers and probe sets were evaluated, and clustering analysis of corresponding amplicon sequences was carried out. Results: From the 1164 representative SARS-CoV-2 Omicron VoC BA.4 sublineage genomes analyzed, a substitution in the first five nucleotides (C to T) of the amplicon's 3' end were observed in all samples resulting in 0% sensitivity for assays HKUnivRdRp/Hel (mismatch in reverse primer) and CoremCharite N (mismatch in both forward and reverse primers). Due to a mismatch in the forward primer's 5' end (3-nucleotide substitution, GGG to AAC), the sensitivity of ChinaCDC N assay was at 0.69%. The 10 nucleotide mismatches in the reverse primer resulted in 0.09% sensitivity for Omicron sublineage BA.4 for Thai N assay. Of the 1926 BA.5 sublineage genomes, HKUnivRdRp/Hel assay also had 0% sensitivity. A sensitivity of 3.06% was observed for ChinaCDC N assay because of a mismatch in the forward primer's 5' end (3-nucleotide substitution, GGG to AAC). Similarly, due to the 10 nucleotide mismatches in the reverse primer, Thai N assay's sensitivity was low at 0.21% for sublineage BA.5. Further, 8 assays for BA.4 sublineage retained high sensitivity (more than 97%) and 9 assays for BA.5 sublineage retained more than 99% sensitivity. Conclusion: We observed four assays (HKUnivRdRp/Hel, ChinaCDC N, Thai N, CoremCharite N) that could potentially result in false negative results for SARS-CoV-2 Omicron VoCs BA.4 and BA.5 sublineages. Interestingly, CoremCharite N had 0% sensitivity for Omicron Voc BA.4 but 99.53% sensitivity for BA.5. In addition, 66.67% of the assays for BA.4 sublineage and 75% of the assays for BA.5 sublineage retained high sensitivity. Further, amplicon clustering and additional substitutions analysis along with sensitivity analysis could be used for modification and development of RT-qPCR assays for detecting SARS-CoV-2 Omicron VoC sublineages. This article is protected by copyright. All rights reserved

Diversity, Phylogeny and Antagonistic Activity of Fungal Endophytes Associated with Endemic Species of Cycas (Cycadales) in China

The culture-based approach was used to characterize the fungal endophytes associated with the coralloid roots of the endemic Cycas debaoensis and Cycas fairylakea from various population sites in China. We aim to determine if the assemblages of fungal endophytes inside these endemic plant hosts are distinct and could be explored for bioprospecting. The isolation method yielded a total of 284 culturable fungal strains. Identification based on the analysis of the internal transcribed spacer (ITS) rDNA showed that they belonged to two phyla, five classes, eight orders and 22 families. At least 33 known genera and 62 different species were confirmed based on >97% ITS sequence similarity. The most frequent and observed core taxa in the two host species regardless of their population origin were Talaromyces, Penicillium, Fusarium, Pochonia and Gliocladiopsis. Seventy percent was a rare component of the fungal communities with only one or two recorded isolates. Contrary to common notions, diversity and fungal richness were significantly higher in C. debaoensis and C. fairylakea collected from a botanical garden, while the lowest was observed in C. debaoensis from a natural habitat; this provides evidence that garden management, and to a minor extent, ex-situ conservation practice, could influence fungal endophyte communities. We further selected nineteen fungal isolates and screened for their antagonistic activities via a co-cultivation approach against the phytopathogens, Diaporthe sp. and Colletotrichum sp. Among these, five isolates with high ITS similarity matches with Hypoxylon vinosupulvinatum (GD019, 99.61%), Penicillium sp. (BD022, 100%), Penicillifer diparietisporus (GD008, 99.46%), Clonostachys rogersoniana (BF024, 99.46%) and C. rosea (BF011, 99.1%), which showed exceptional antagonistic activities against the phytopathogenic fungi with a significant inhibition rate of 70–80%. Taken together, our data presented the first and most comprehensive molecular work on culturable fungal endophytes associated with the coralloid roots of cycads. Our study also demonstrated that about 5% of fungal endophytes were not detected by the high-throughput sequencing approach, implying the equal importance of a culture-dependent approach to study fungal communities of cycads. We further highlighted the potential role of endemic and rare plants to discover and isolate unique plant-associated fungal taxa with excellent biocontrol properties

Platelet distribution width (PDW) as a significant correlate of COVID-19 infection severity and mortality

: SARS-CoV-2 infection may cause a wide spectrum of symptoms, from asymptomatic, to mild respiratory symptoms and life-threatening sepsis. Among the clinical laboratory biomarkers analyzed during COVID-19 pandemic, platelet indices have raised great interest, due to the critical involvement of platelets in COVID-19-related thromboinflammation. Through an electronic literature search on MEDLINE, CINAHL, PubMed, EMBASE, Web of Science, and preprint servers we performed and updated a systematic review aimed at providing a detailed analysis of studies addressing the potential clinical utility of platelet distribution width, platelet distribution width (PDW), in laboratory medicine, exploring the possible association between increased PDW levels, disease severity, and mortality in COVID-19. Our systematic review revealed a wide heterogeneity of COVID-19 cohorts examined and a lack of homogenous expression of platelet indices. We found that 75 % of studies reported significantly elevated PDW values in COVID-19 infected cohorts compared to healthy/non-COVID-19 controls, and 40 % of studies reported that patients with severe COVID-19 showed increased PDW values than those with less-than-severe illness. Interestingly, 71.4 % of studies demonstrated significant increased PDW values in non survivors vs. survivors. Overall, these results suggest that platelets are critically involved as major players in the process of immunothrombosis in COVID-19, and platelet reactivity and morphofunctional alterations are mirrored by PDW, as indicator of platelet heterogeneity. Our results confirm that the use of PDW as prognostic biomarkers of COVID-19 sepsis still remains debated due to the limited number of studies to draw a conclusion, but new opportunities to investigate the crucial role of platelets in thrombo-inflammation are warranted

Platelet distribution width (PDW) as a significant correlate of COVID-19 infection severity and mortality

: SARS-CoV-2 infection may cause a wide spectrum of symptoms, from asymptomatic, to mild respiratory symptoms and life-threatening sepsis. Among the clinical laboratory biomarkers analyzed during COVID-19 pandemic, platelet indices have raised great interest, due to the critical involvement of platelets in COVID-19-related thromboinflammation. Through an electronic literature search on MEDLINE, CINAHL, PubMed, EMBASE, Web of Science, and preprint servers we performed and updated a systematic review aimed at providing a detailed analysis of studies addressing the potential clinical utility of platelet distribution width, platelet distribution width (PDW), in laboratory medicine, exploring the possible association between increased PDW levels, disease severity, and mortality in COVID-19. Our systematic review revealed a wide heterogeneity of COVID-19 cohorts examined and a lack of homogenous expression of platelet indices. We found that 75 % of studies reported significantly elevated PDW values in COVID-19 infected cohorts compared to healthy/non-COVID-19 controls, and 40 % of studies reported that patients with severe COVID-19 showed increased PDW values than those with less-than-severe illness. Interestingly, 71.4 % of studies demonstrated significant increased PDW values in non survivors vs. survivors. Overall, these results suggest that platelets are critically involved as major players in the process of immunothrombosis in COVID-19, and platelet reactivity and morphofunctional alterations are mirrored by PDW, as indicator of platelet heterogeneity. Our results confirm that the use of PDW as prognostic biomarkers of COVID-19 sepsis still remains debated due to the limited number of studies to draw a conclusion, but new opportunities to investigate the crucial role of platelets in thrombo-inflammation are warranted

Comparative Study on the Antimicrobial Activities and Metabolic Profiles of Five <i>Usnea</i> Species from the Philippines

The rapid emergence of resistant bacteria is occurring worldwide, endangering the efficacy of antibiotics. Hence, there is a need to search for new sources of antibiotics that either exhibit novel structures or express a new mechanism of action. The lichen Usnea, with its wide range of unique, biologically potent secondary metabolites, may solve this problem. In this study, Usnea species were collected in the Northern Philippines, identified through combined morphological and biochemical characterization, and tested for antimicrobial activities against the multidrug-resistant ESKAPE pathogens, i.e., Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae, two standard antibiotic-sensitive test bacteria, and a yeast. A total of 46 lichen specimens were collected and later identified as Usnea baileyi (10), U. diffracta (10), U. glabrata (12), U. longissima (4), and U. rubicunda (10). The results show that the crude extracts of the Usnea species exhibited promising in vitro inhibitory activities against standard antibiotic-sensitive (E. faecalis ATCC 29212) and multidrug-resistant (methicillin-resistant S. aureus and E. faecalis) Gram-positive bacteria. Additionally, lichen compounds of representative specimens per species were identified and profiled using thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The detection of lichen acids (LA) via HPLC showed the presence of 24 peaks of lichen acids. TLC-bioautography identified the bioactive lichen acids as alectronic acid, connorstictic acid, consalazinic acid, diffractaic acid, echinocarpic acid, erythrin acid, galbinic acid, hypoconstictic acid, hyposalazinic acid, hypostictic acid, lobaric acid, menegazzaic acid, micareic acid, pannarin, salazinic acid, stictic acid, and usnic acid. Our study highlighted the wide spectrum of opportunities for using lichens for the discovery of potential antimicrobial agents

Butachlor biodegradation potential of fungi isolated from submerged wood and surface water collected in Taal Lake, Philippines

Taal Lake in the Philippines is a hotspot of diverse organisms but also faces a great threat from toxic agrochemical contaminants. Specifically, fungi are promising bioremediation agents to degrade harmful pollutants in the environment; hence the documentation and identification of these isolates are crucial. This study aims to determine the occurrence of fungi from submerged woods and surface waters in Taal Lake and assess their biodegradation potential against butachlor, a widely used pesticide in the Philippines. The isolated fungi were identified by using morphological and/or molecular methods, and the occurrence of each species was recorded. The ability of selected fungal isolates to degrade butachlor was also determined. A total of 28 morphospecies belonging to 20 fungal genera were recorded. Of these, eight fungal isolates grew on a chemically defined medium with up to 100 ppm butachlor. Two fungal isolates identified by molecular methods as Neodeightonia subglobosa IFM 63572 and Sclerotium hydrophilum IFM 63573 effectively utilized and potentially degraded butachlor as their sole carbon source as evident in the increased mycelial biomass (up to 0.449 g/L increase for N. subglobosa IFM 63572 and 0.214 g/L for S. hydrophilum IFM 63573) and decreased butachlor concentration (up to 94.68% reduction for N. subglobosa IFM 63572 and 89.64% for S. hydrophilum IFM 63573) after five days of incubation. Mycelial mat was more effective in degrading butachlor than mycelial balls. This study showed the presence of fungi from submerged woods and surface waters in Taal Lake and their potential application in the biodegradation of butachlor

Combinatorial library screening of quinadoline B derivatives against SARS-CoV-2 RNA-dependent RNA polymerase

The unprecedented global health threat of SARS-CoV-2 has sparked a continued interest to discover novel anti-COVID-19 agents. To this end, we present here a computer-based protocol for identifying potential compounds targeting RNA-dependent RNA polymerase (RdRp). Starting from our previous study in which, by a virtual screening campaign, we identified a fumiquinazolinone alkaloid quinadoline B (Q3), an antiviral fungal metabolite with significant activity against SARS-CoV-2 RdRp, we applied an in silico combinatorial methodologies for generating and screening a library of anti-SARS-CoV-2 candidates with strong in silico affinity for RdRp. For this study, the quinadoline pharmacophore was subjected to structural iteration obtaining a Q3-focused library of over 900,000 unique structures. This chemical library was explored to identify binders of RdRp with greater affinity with respect to the starting compound Q3. Coupling this approach with the evaluation of physchem profile, we found 26 compounds with significant affinities for the RdRp binding site. Moreover, top-ranked compounds were submitted to molecular dynamics to evaluate the stability of the systems during a selected time, and for deeply investigating the binding mode of the most promising derivatives. Among the generated structures, five compounds, obtained by inserting nucleotide-like scaffolds (1, 2, and 5), heterocyclic thiazolyl benzamide moiety (compound 3), and a peptide residue (compound 4), exhibited enhanced binding affinity for SARS-CoV-2 RdRp, deserving further investigation as possible antiviral agents. Remarkably, the presented in silico procedure provides a useful computational procedure for hit-to-lead optimization, having implications in anti-SARS-CoV-2 drug discovery and in general in the drug optimization process

Virtual Combinatorial Library Screening of Quinadoline B Derivatives against SARS-CoV-2 RNA-Dependent RNA Polymerase

The unprecedented global health threat of SARS-CoV-2 has sparked a continued interest in discovering novel anti-COVID-19 agents. To this end, we present here a computer-based protocol for identifying potential compounds targeting RNA-dependent RNA polymerase (RdRp). Starting from our previous study wherein, using a virtual screening campaign, we identified a fumiquinazolinone alkaloid quinadoline B (Q3), an antiviral fungal metabolite with significant activity against SARS-CoV-2 RdRp, we applied in silico combinatorial methodologies for generating and screening a library of anti-SARS-CoV-2 candidates with strong in silico affinity for RdRp. For this study, the quinadoline pharmacophore was subjected to structural iteration, obtaining a Q3-focused library of over 900,000 unique structures. This chemical library was explored to identify binders of RdRp with greater affinity with respect to the starting compound Q3. Coupling this approach with the evaluation of physchem profile, we found 26 compounds with significant affinities for the RdRp binding site. Moreover, top-ranked compounds were submitted to molecular dynamics to evaluate the stability of the systems during a selected time, and to deeply investigate the binding mode of the most promising derivatives. Among the generated structures, five compounds, obtained by inserting nucleotide-like scaffolds (1, 2, and 5), heterocyclic thiazolyl benzamide moiety (compound 3), and a peptide residue (compound 4), exhibited enhanced binding affinity for SARS-CoV-2 RdRp, deserving further investigation as possible antiviral agents. Remarkably, the presented in silico procedure provides a useful computational procedure for hit-to-lead optimization, having implications in anti-SARS-CoV-2 drug discovery and in general in the drug optimization process