T-RFPred: a nucleotide sequence size prediction tool for microbial community description based on terminal-restriction fragment length polymorphism chromatograms

<p>Abstract</p> <p>Background</p> <p>Terminal-Restriction Fragment Length Polymorphism (T-RFLP) is a technique used to analyze complex microbial communities. It allows for the quantification of unique or numerically dominant phylotypes in amplicon pools and it has been used primarily for comparisons between different communities. T-RFPred, Terminal-Restriction Fragment Prediction, was developed to identify and assign taxonomic information to chromatogram peaks of a T-RFLP fingerprint for a more comprehensive description of microbial communities. The program estimates the expected fragment size of representative 16S rRNA gene sequences (either from a complementary clone library or from public databases) for a given primer and restriction enzyme(s) and provides candidate taxonomic assignments.</p> <p>Results</p> <p>To show the accuracy of the program, T-RFLP profiles of a marine bacterial community were described using artificial bacterioplankton clone libraries of sequences obtained from public databases. For all valid chromatogram peaks, a phylogenetic group could be assigned.</p> <p>Conclusions</p> <p>T-RFPred offers enhanced functionality of T-RFLP profile analysis over current available programs. In particular, it circumvents the need for full-length 16S rRNA gene sequences during taxonomic assignments of T-RF peaks. Thus, large 16S rRNA gene datasets from environmental studies, including metagenomes, or public databases can be used as the reference set. Furthermore, T-RFPred is useful in experimental design for the selection of primers as well as the type and number of restriction enzymes that will yield informative chromatograms from natural microbial communities.</p

Steroidal Saponins from Furcraea hexapetala Leaves and Their Phytotoxic Activity

Four new steroidal saponins (1−4) along with 13 known saponins were isolated from the leaves of Furcraea hexapetala. The new compounds were identified as (20R,22R,25R)-3β-hydroxy-5α-spirostan-12-one 3-O-{α-Lrhamnopyranosyl-(1→4)-O-β-D-glucopyranosyl-(1→3)-O-[β-D-glucopyranosyl-(1→3)-O-β-D-glucopyranosyl-(1→2)]-O-β-D-glucopyranosyl-(1→4)-O-β-D-galactopyranoside} (1), (25R)-3β-hydroxy-5α-spirost-20(21)-en-12-one 3-O-{α-Lrhamnopyranosyl-(1→4)-O-β-D-glucopyranosyl-(1→3)-O-[β-D-glucopyranosyl-(1→3)-O-β-D-glucopyranosyl-(1→2)]-O-β-D-glucopyranosyl-(1→4)-O-β-D-galactopyranoside} (2), (25R)-5α-spirostan-3β-ol 3-O-{β-D-glucopyranosyl-(1→2)-O-β-D-glucopyranosyl-(1→2)-O-β-D-glucopyranosyl-(1→4)-O-β-D-galactopyranoside} (3), and (25R)-5β-spirostan-3β-ol 3-O-{β-D-glucopyranosyl-(1→6)-O-β-D-galactopyranoside} (4) by spectroscopic analysis, including one- and two-dimensional NMR techniques, mass spectrometry, and chemical methods. The phytotoxicity of the isolated compounds against the standard target species Lactuca sativa was evaluated. Structure−activity relationships for these compounds with respect to phytotoxic effects are discussed

Bioactive steroidal saponins from Agave offoyana flowers

Bioguided studies of flowers of Agave offoyana allowed the isolation of five steroidal saponins never described previously, Magueyosides A–E (1–5), along with six known steroidal saponins (6–11). The structures of compounds were determined as (25R)-spirost-5-en-2a,3b-diol-12-one 3-O-{b-D-xylopyranosyl-(1-3)-O-b-D-glucopyranosyl-(1-2)-O-[b-D-xylopyranosyl-(1-3)]-O-b-D-glucopyranosyl-( 1-4)-O-b-D-galactopyranoside} (1), (25R)-spirost-5-en-2a,3b-diol-12-one 3-O-{b-D-glucopyranosyl-(1-2)-O-[b-D-xylopyranosyl-(1-3)]-O-b-D-glucopyranosyl-(1-4)-O-b-D galactopyranoside} (2), (25R)-spirost-5-en-2a,3b,12b-triol 3-O-{b-D-glucopyranosyl-(1-2)-O-[b-D-xylopyranosyl-(1-3)]- O-b-D-glucopyranosyl-(1-4)-O-b-D-galactopyranoside} (3), (25R)-5a-spirostan-2a,3b-diol-12-one 3-O-{b-D-xylopyranosyl-(1-3)-O-b-D-glucopyranosyl-(1-2)-O-[b-D-xylopyranosyl-(1-3)]-O-b-D-glucopyranosyl-(1-4)-O-b-D-galactopyranoside} (4), and (25R)-5a-spirostan-2a,3b-diol-9(11)-en-12-one 3-O-{b-D-xylopyranosyl-(1-3)-O-b-D-glucopyranosyl-(1-2)-O-[b-D-xylopyranosyl-(1-3)]-O-b-D-glucopyranosyl-( 1-4)-O-b-D-galactopyranoside} (5), by comprehensive spectroscopic analysis, including one- and two-dimensional NMR techniques, mass spectrometry and chemical methods. The bioactivities of the isolated compounds on the standard target species Lactuca sativa were evaluated. A dosedependent phytotoxicity and low dose stimulation were observed

Phytotoxic steroidal saponins from Agave offoyana leaves

A bioassay-guided fractionation of Agave offoyana leaves led to the isolation of five steroidal saponins (1–5) along with six known saponins (6–11). The compounds were identified as (25R)-spirost-5-en-2α,3β-diol-12-one 3-O-{α-L-rhamnopyranosyl-(1→3)-O-β-D-glucopyranosyl-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-O-β-D-glucopyranosyl-(1→4)-O-β-D-galactopyranoside} (1), (25R)-spirost-5-en-3β-ol-12-one 3-O-{α-L-rhamnopyranosyl-(1→3)-O-β-D-glucopyranosyl-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-O-β-D-glu copyranosyl-(1→4)-O-β-D-galactopyranoside} (2), (25R)-spirost-5-en-3β-ol-12-one 3-O-{β-D-xylopyrano syl-(1→3)-O-β-D-glucopyranosyl-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-O-β-D-glucopyranosyl-(1→4)-O-β -D-galactopyranoside} (3), (25R)-26-O-β-D-glucopyranosylfurost-5-en-3β,22α,26-triol-12-one 3-O- {α-L-rhamnopyranosyl-(1→3)-O-β-D-glucopyranosyl-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-O-β-D-glucopyrano syl-(1→4)-O-β-D-galactopyranoside} (4) and (25R)-26-O-β-D-glucopyranosylfurost-5-en-3β,22α,26-triol- 12-one 3-O-{β-D-xylopyranosyl-(1→3)-O-β-D-glucopyranosyl-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-O-β- D-glucopyranosyl-(1→4)-O-β-D-galactopyranoside} (5) by comprehensive spectroscopic analysis, including one- and two-dimensional NMR techniques, mass spectrometry and chemical methods. The phytotoxicity of the isolated compounds on the standard target species Lactuca sativa was evaluated

Ecology of marine Bacteroidetes: a comparative genomics approach

5th Congress of European Microbiologists (FEMS 2013), 21-25 july 2013, Leipzig, Germany.-- 1 pagePeer Reviewe

Extending Graph (Discrete) Derivative Descriptors to N-Tuple Atom-Relations

In the present manuscript, an extension of the previously defined Graph Derivative Indices (GDIs) is discussed. To achieve this objective, the concept of a hypermatrix, conceived from the calculation of the frequencies of triple and quadruple atom relations in a set of connected sub-graphs, is introduced. This set of subgraphs is generated following a predefined criterion, known as the event (S), being in this particular case the connectivity among atoms. The triple and quadruple relations frequency matrices serve as a basis for the computation of triple and quadruple discrete derivative indices, respectively. The GDIs are implemented in a computational program denominated DIVATI (acronym for DIscrete DeriVAtive Type Indices), a module of TOMOCOMD-CARDD program. Shannon‟s entropy-based variability analysis demonstrates that the GDIs show major variability than others indices used in QSAR/QSPR researches. In addition, it can be appreciated when the indices are extended over n-elements from the graph, its quality increases, principally when they are used in a combined way. QSPR modeling of the physicochemical properties Log P and Log K of the 2-furylethylenes derivatives reveals that the GDIs obtained using the tripleand quadruple matrix approaches yield superior performance to the duplex matrix approach. Moreover, the statistical parameters for models obtained with the GDI method are superior to those reported in the literature by using other methods. It can therefore be suggested that the GDI method, seem to be a promissory tool to reckon on in QSAR/QSPR studies, virtual screening of compound datasets and similarity/dissimilarity evaluations

Persistent SARS-CoV-2 PCR Positivity Despite Anti-viral Treatment in Immunodeficient Patients

PURPOSE: COVID-19 infection in immunodeficient individuals can result in chronically poor health, persistent or relapsing SARS-CoV-2 PCR positivity, and long-term infectious potential. While clinical trials have demonstrated promising outcomes using anti-SARS-CoV-2 medicines in immunocompetent hosts, their ability to achieve sustained viral clearance in immunodeficient patients remains unknown. We therefore aimed to study long-term virological outcomes in patients treated at our centre. METHODS: We followed up immunocompromised inpatients treated with casirivimab-imdevimab (Ronapreve) between September and December 2021, and immunocompromised patients who received sotrovimab, molnupiravir, nirmatrelvir/ritonavir (Paxlovid), or no treatment from December 2021 to March 2022. Nasopharyngeal swab and sputum samples were obtained either in hospital or in the community until sustained viral clearance, defined as 3 consecutive negative PCR samples, was achieved. Positive samples were sequenced and analysed for mutations of interest. RESULTS: We observed sustained viral clearance in 71 of 103 patients, none of whom died. Of the 32/103 patients where sustained clearance was not confirmed, 6 died (between 2 and 34 days from treatment). Notably, we observed 25 cases of sputum positivity despite negative nasopharyngeal swab samples, as well as recurrence of SARS-CoV-2 positivity following a negative sample in 12 cases. Patients were then divided into those who cleared within 28 days and those with PCR positivity beyond 28 days. We noted lower B cell counts in the group with persistent PCR positivity (mean (SD) 0.06 (0.10) ×109/L vs 0.22 (0.28) ×109/L, p = 0.015) as well as lower IgA (median (IQR) 0.00 (0.00-0.15) g/L vs 0.40 (0.00-0.95) g/L, p = 0.001) and IgM (median (IQR) 0.05 (0.00-0.28) g/L vs 0.35 (0.10-1.10) g/L, p = 0.005). No differences were seen in CD4+ or CD8+ T cell counts. Antiviral treatment did not impact risk of persistent PCR positivity. CONCLUSION: Persistent SARS-CoV-2 PCR positivity is common among immunodeficient individuals, especially those with antibody deficiencies, regardless of anti-viral treatment. Peripheral B cell count and serum IgA and IgM levels are predictors of viral persistence

An Influenza A/H1N1/2009 Hemagglutinin Vaccine Produced in Escherichia coli

The A/H1N1/2009 influenza pandemic made evident the need for faster and higher-yield methods for the production of influenza vaccines. Platforms based on virus culture in mammalian or insect cells are currently under investigation. Alternatively, expression of fragments of the hemagglutinin (HA) protein in prokaryotic systems can potentially be the most efficacious strategy for the manufacture of large quantities of influenza vaccine in a short period of time. Despite experimental evidence on the immunogenic potential of HA protein constructs expressed in bacteria, it is still generally accepted that glycosylation should be a requirement for vaccine efficacy.We expressed the globular HA receptor binding domain, referred to here as HA(63-286)-RBD, of the influenza A/H1N1/2009 virus in Escherichia coli using a simple, robust and scalable process. The recombinant protein was refolded and purified from the insoluble fraction of the cellular lysate as a single species. Recombinant HA(63-286)-RBD appears to be properly folded, as shown by analytical ultracentrifugation and bio-recognition assays. It binds specifically to serum antibodies from influenza A/H1N1/2009 patients and was found to be immunogenic, to be capable of triggering the production of neutralizing antibodies, and to have protective activity in the ferret model.Projections based on our production/purification data indicate that this strategy could yield up to half a billion doses of vaccine per month in a medium-scale pharmaceutical production facility equipped for bacterial culture. Also, our findings demonstrate that glycosylation is not a mandatory requirement for influenza vaccine efficacy