A cry for kelp: Evidence for polyphenolic inhibition of Oxford Nanopore sequencing of brown algae

\ua9 2024 The Author(s). Journal of Phycology published by Wiley Periodicals LLC on behalf of Phycological Society of America.Genomic resources have yielded unprecedented insights into ecological and evolutionary processes, not to mention their importance in economic and conservation management of specific organisms. However, the field of macroalgal genomics is hampered by difficulties in the isolation of suitable DNA. Even when DNA that appears high quality by standard metrics has been isolated, such samples may not perform well during the sequencing process. We here have compared Oxford Nanopore long-read sequencing results for three species of macroalgae to those of nonmacroalgal species and determined that when using macroalgal samples, sequencing activity declined rapidly, resulting in reduced sequencing yield. Chemical analysis of macroalgal DNA that would be considered suitable for sequencing revealed that DNA derived from dried macroalgae was enriched for polyphenol–DNA adducts (DNA with large polyphenols chemically attached to it), which may have led to sequencing inhibition. Of note, we observed the strongest evidence of sequencing inhibition and reduced sequence output when using samples dried using silica gel—suggesting that such storage approaches may not be appropriate for samples destined for Oxford Nanopore sequencing. Our findings have wide-ranging implications for the generation of genomic resources from macroalgae and suggest a need to develop new storage methods that are more amenable to Oxford Nanopore sequencing or to use fresh flash-frozen tissue wherever possible for genome sequencing

Uncoupling Molecular Testing for SARS-CoV-2 From International Supply Chains

The rapid global rise of COVID-19 from late 2019 caught major manufacturers of RT-qPCR reagents by surprise and threw into sharp focus the heavy reliance of molecular diagnostic providers on a handful of reagent suppliers. In addition, lockdown and transport bans, necessarily imposed to contain disease spread, put pressure on global supply lines with freight volumes severely restricted. These issues were acutely felt in New Zealand, an island nation located at the end of most supply lines. This led New Zealand scientists to pose the hypothetical question: in a doomsday scenario where access to COVID-19 RT-qPCR reagents became unavailable, would New Zealand possess the expertise and infrastructure to make its own reagents onshore? In this work we describe a review of New Zealand's COVID-19 test requirements, bring together local experts and resources to make all reagents for the RT-qPCR process, and create a COVID-19 diagnostic assay referred to as HomeBrew (HB) RT-qPCR from onshore synthesized components. This one-step RT-qPCR assay was evaluated using clinical samples and shown to be comparable to a commercial COVID-19 assay. Through this work we show New Zealand has both the expertise and, with sufficient lead time and forward planning, infrastructure capacity to meet reagent supply challenges if they were ever to emerge.fals

A classification of diabetic foot infections using ICD-9-CM codes: application to a large computerized medical database

<p>Abstract</p> <p>Background</p> <p>Diabetic foot infections are common, serious, and varied. Diagnostic and treatment strategies are correspondingly diverse. It is unclear how patients are managed in actual practice and how outcomes might be improved. Clarification will require study of large numbers of patients, such as are available in medical databases. We have developed and evaluated a system for identifying and classifying diabetic foot infections that can be used for this purpose.</p> <p>Methods</p> <p>We used the (VA) Diabetes Epidemiology Cohorts (DEpiC) database to conduct a retrospective observational study of patients with diabetic foot infections. DEpiC contains computerized VA and Medicare patient-level data for patients with diabetes since 1998. We determined which ICD-9-CM codes served to identify patients with different types of diabetic foot infections and ranked them in declining order of severity: Gangrene, Osteomyelitis, Ulcer, Foot cellulitis/abscess, Toe cellulitis/abscess, Paronychia. We evaluated our classification by examining its relationship to patient characteristics, diagnostic procedures, treatments given, and medical outcomes.</p> <p>Results</p> <p>There were 61,007 patients with foot infections, of which 42,063 were classifiable into one of our predefined groups. The different types of infection were related to expected patient characteristics, diagnostic procedures, treatments, and outcomes. Our severity ranking showed a monotonic relationship to hospital length of stay, amputation rate, transition to long-term care, and mortality.</p> <p>Conclusions</p> <p>We have developed a classification system for patients with diabetic foot infections that is expressly designed for use with large, computerized, ICD-9-CM coded administrative medical databases. It provides a framework that can be used to conduct observational studies of large numbers of patients in order to examine treatment variation and patient outcomes, including the effect of new management strategies, implementation of practice guidelines, and quality improvement initiatives.</p

Protein profiling of the dimorphic, pathogenic fungus, Penicillium marneffei

<p>Abstract</p> <p>Background</p> <p><it>Penicillium marneffei </it>is a pathogenic fungus that afflicts immunocompromised individuals having lived or traveled in Southeast Asia. This species is unique in that it is the only dimorphic member of the genus. Dimorphism results from a process, termed phase transition, which is regulated by temperature of incubation. At room temperature, the fungus grows filamentously (mould phase), but at body temperature (37°C), a uninucleate yeast form develops that reproduces by fission. Formation of the yeast phase appears to be a requisite for pathogenicity. To date, no genes have been identified in <it>P. marneffei </it>that strictly induce mould-to-yeast phase conversion. In an effort to help identify potential gene products associated with morphogenesis, protein profiles were generated from the yeast and mould phases of <it>P. marneffei</it>.</p> <p>Results</p> <p>Whole cell proteins from the early stages of mould and yeast development in <it>P. marneffei </it>were resolved by two-dimensional gel electrophoresis. Selected proteins were recovered and sequenced by capillary-liquid chromatography-nanospray tandem mass spectrometry. Putative identifications were derived by searching available databases for homologous fungal sequences. Proteins found common to both mould and yeast phases included the signal transduction proteins cyclophilin and a RACK1-like ortholog, as well as those related to general metabolism, energy production, and protection from oxygen radicals. Many of the mould-specific proteins identified possessed similar functions. By comparison, proteins exhibiting increased expression during development of the parasitic yeast phase comprised those involved in heat-shock responses, general metabolism, and cell-wall biosynthesis, as well as a small GTPase that regulates nuclear membrane transport and mitotic processes in fungi. The cognate gene encoding the latter protein, designated <it>RanA</it>, was subsequently cloned and characterized. The <it>P. marneffei </it>RanA protein sequence, which contained the signature motif of Ran-GTPases, exhibited 90% homology to homologous <it>Aspergillus </it>proteins.</p> <p>Conclusion</p> <p>This study clearly demonstrates the utility of proteomic approaches to studying dimorphism in <it>P. marneffei</it>. Moreover, this strategy complements and extends current genetic methodologies directed towards understanding the molecular mechanisms of phase transition. Finally, the documented increased levels of RanA expression suggest that cellular development in this fungus involves additional signaling mechanisms than have been previously described in <it>P. marneffei</it>.</p

ADDITIVE EFFECTS OF MILD DEHYDRATION AND HYPERTHERMIA ON MOOD IN FEMALES

Jenna M. Burchfield, Matthew S. Ganio, Brendon P. McDermott, Nicole E. Moyen, Cory L. Butts, Keeley Treece & Matthew A. Tucker Human Performance Laboratory, Department of Health, Human Performance, and Recreation, University of Arkansas, Fayetteville, Arkansas INTRODUCTION: Information regarding effects of dehydration (DY) and heat stress on mood and perceptual measures is controversial since the two conditions could confound one another. PURPOSE: To assess the independent and combined effects of mild DY through 24-h fluid restriction and heat stress via passive heating on mood, thirst, and thermal sensation in females. METHODS: Eight healthy females (24.3 ± 4.6 y, 162.4 ± 4.2 cm, 77.3 ± 12.9 kg) volunteered in two randomized, repeated-measures trials, both involving passive heating to a 1.0°C core temperature (TC) increase from baseline. 24-h prior to heat stress, females either remained euhydrated (EU) or became DY via fluid restriction. Upon arrival, percent body mass (BM) change was calculated by comparing their nude BM to a 3-day baseline BM record. EU individuals received fluid during heating to maintain euhydration. Percent BM change and blood (osmolality) and urine (osmolality and specific gravity) biomarkers were measured prior to and throughout testing. Thirst, thermal sensation, and mood (Brunel Mood Scale; BRUMS) were assessed via questionnaires before heat stress, at 0.5ºC, and 1.0ºC increases in core temperature. RESULTS: EU started the heating euhydrated according to BM change from baseline (-0.4±0.9%), urine specific gravity (USG; 1.009 ±0.007), urine osmolality (UOSM; 331±241 mOsm/kgH2O), and plasma osmolality (POSM; 286±4 mOsm/kgH2O). DY participants started heat stress with a BM change from baseline of -1.2±0.4%, USG of 1.027±0.004, UOSM of 1058±130 mOsm/kgH2O, and POSM of 290±4 mOsm/kgH2O. Despite physiological confirmation of dehydration, there were no differences in mood state, thirst, or thermal sensation between EU and DY at baseline (p \u3e 0.05). Further the effect of heat stress on these variables was similar between groups. Independent of group, thermal sensation significantly increased with heat stress from baseline to 1.0°C TC increase (4.6 ± 0.8 to 6.7 ± 0.3, p \u3c 0.05). However, independent of heat stress, DY subjects had significantly decreased Vigor (41.9 ± 3.6 v 37.3 ± 2.3 arbitrary units p \u3c 0.05) and increased thirst (3.8 ± 0.5 v 6.6 ± 0.5, all p \u3c 0.05). CONCLUSION: These data indicate that DY and heat stress do not additively affect mood or perceptual measures. Although heat stress does not affect mood, emphasis should still be placed on keeping participants hydrated when testing effects of heat stress, as hydration status alone affected Vigor and thirst independently of heating. Heat stress and dehydration were not additive in this setting. Heat stress failed to have an independent affect on mood or thirst measures, but dehydration alone decreased Vigor

A method for simultaneous detection of small and long RNA biotypes by ribodepleted RNA-Seq

RNA sequencing offers unprecedented access to the transcriptome. Key to this is the identification and quantification of many different species of RNA from the same sample at the same time. In this study we describe a novel protocol for simultaneous detection of coding and non-coding transcripts using modifications to the Ion Total RNA-Seq kit v2 protocol, with integration of QIASeq FastSelect rRNA removal kit. We report highly consistent sequencing libraries can be produced from both frozen high integrity mouse hippocampal tissue and the more challenging post-mortem human tissue. Removal of rRNA using FastSelect was extremely efficient, resulting in less than 1.5% rRNA content in the final library. We identified > 30,000 unique transcripts from all samples, including protein-coding genes and many species of non-coding RNA, in biologically-relevant proportions. Furthermore, the normalized sequencing read count for select genes significantly negatively correlated with Ct values from qRT-PCR analysis from the same samples. These results indicate that this protocol accurately and consistently identifies and quantifies a wide variety of transcripts simultaneously. The highly efficient rRNA depletion, coupled with minimized sample handling and without complicated and high-loss size selection protocols, makes this protocol useful to researchers wishing to investigate whole transcriptomes

Education for secondary prevention of foot ulcers in people with diabetes: a randomised controlled trial

Aims/hypothesis This observer-blind, randomised controlled trial was designed to determine the effect of a foot care education programme in the secondary prevention of foot ulcers. Methods People with newly healed foot ulcers attending one of three specialist clinics were allocated to receive either targeted, one-to-one education or usual care, using a computer-generated random allocation sequence that had been prepared in advance but which was concealed from the clinical researcher. The primary outcome was ulcer incidence at 12 months. Secondary outcomes were ulcer incidence at 6 months and incidence of amputation, mood (Hospital Anxiety and Depression Scale) and quality of life (Diabetic Foot Ulcer Scale) at 6 and 12 months. Protective foot care behaviours (Nottingham Assessment of Functional Footcare) were assessed at 12 months. Results There were 87 (mean [SD] age 63.5 [12.1] years) patients in the intervention group and 85 control patients (mean [SD] age 64.9 [10.9] years). The groups were comparable at baseline. No significant differences (p > 0.05) were observed between groups in ulcer incidence at either 6 months (intervention 30%, control 21%) or 12 months (intervention 41%, control 41%). Recommended foot care behaviours at 12 months were better in the intervention than in the control group (p = 0.03), but education had no significant (p > 0.05) effect on mood, quality of life or amputations. Conclusions/interpretation Even though the intervention was associated with improved foot care behaviour, there was no evidence that this programme of targeted education was associated with clinical benefit in this population when compared with usual care. The usefulness and optimal delivery of education to such a high-risk group requires further evaluation