Finding needles in haystacks:Linking scientific names, reference specimens and molecular data for Fungi

DNA phylogenetic comparisons have shown that morphology-based species recognition often underestimates fungal diversity. Therefore, the need for accurate DNA sequence data, tied to both correct taxonomic names and clearly annotated specimen data, has never been greater. Furthermore, the growing number of molecular ecology and microbiome projects using high-throughput sequencing require fast and effective methods for en masse species assignments. In this article, we focus on selecting and re-annotating a set of marker reference sequences that represent each currently accepted order of Fungi. The particular focus is on sequences from the internal transcribed spacer region in the nuclear ribosomal cistron, derived from type specimens and/or ex-type cultures. Reannotated and verified sequences were deposited in a curated public database at the National Center for Biotechnology Information (NCBI), namely the RefSeq Targeted Loci (RTL) database, and will be visible during routine sequence similarity searches with NR_prefixed accession numbers. A set of standards and protocols is proposed to improve the data quality of new sequences, and we suggest how type and other reference sequences can be used to improve identification of Fungi.The Intramural Research Programs of the National Center for Biotechnology Information, National Library of Medicine and the National Human Genome Research Institute, both at the National Institutes of Health.http://www.ncbi.nlm.nih.gov/bioproject/PRJNA177353am201

Finding needles in haystacks : linking scientific names, reference specimens and molecular data for Fungi

DNA phylogenetic comparisons have shown that morphology-based species recognition often underestimates fungal diversity. Therefore, the need for accurate DNA sequence data, tied to both correct taxonomic names and clearly annotated specimen data, has never been greater. Furthermore, the growing number of molecular ecology and microbiome projects using high-throughput sequencing require fast and effective methods for en masse species assignments. In this article, we focus on selecting and re-annotating a set of marker reference sequences that represent each currently accepted order of Fungi. The particular focus is on sequences from the internal transcribed spacer region in the nuclear ribosomal cistron, derived from type specimens and/or ex-type cultures. Reannotated and verified sequences were deposited in a curated public database at the National Center for Biotechnology Information (NCBI), namely the RefSeq Targeted Loci (RTL) database, and will be visible during routine sequence similarity searches with NR_prefixed accession numbers. A set of standards and protocols is proposed to improve the data quality of new sequences, and we suggest how type and other reference sequences can be used to improve identification of Fungi.The Intramural Research Programs of the National Center for Biotechnology Information, National Library of Medicine and the National Human Genome Research Institute, both at the National Institutes of Health.http://www.ncbi.nlm.nih.gov/bioproject/PRJNA177353am201

COVID-19 symptoms at hospital admission vary with age and sex: results from the ISARIC prospective multinational observational study

Background: The ISARIC prospective multinational observational study is the largest cohort of hospitalized patients with COVID-19. We present relationships of age, sex, and nationality to presenting symptoms. Methods: International, prospective observational study of 60 109 hospitalized symptomatic patients with laboratory-confirmed COVID-19 recruited from 43 countries between 30 January and 3 August 2020. Logistic regression was performed to evaluate relationships of age and sex to published COVID-19 case definitions and the most commonly reported symptoms. Results: ‘Typical’ symptoms of fever (69%), cough (68%) and shortness of breath (66%) were the most commonly reported. 92% of patients experienced at least one of these. Prevalence of typical symptoms was greatest in 30- to 60-year-olds (respectively 80, 79, 69%; at least one 95%). They were reported less frequently in children (≤ 18 years: 69, 48, 23; 85%), older adults (≥ 70 years: 61, 62, 65; 90%), and women (66, 66, 64; 90%; vs. men 71, 70, 67; 93%, each P < 0.001). The most common atypical presentations under 60 years of age were nausea and vomiting and abdominal pain, and over 60 years was confusion. Regression models showed significant differences in symptoms with sex, age and country. Interpretation: This international collaboration has allowed us to report reliable symptom data from the largest cohort of patients admitted to hospital with COVID-19. Adults over 60 and children admitted to hospital with COVID-19 are less likely to present with typical symptoms. Nausea and vomiting are common atypical presentations under 30 years. Confusion is a frequent atypical presentation of COVID-19 in adults over 60 years. Women are less likely to experience typical symptoms than men

Successful Use of WALANT in Local and Regional Soft Tissue Flaps: A Case Series

Background:. The wide awake local anesthesia no tourniquet (WALANT) technique has been proven to be safe and effective for upper extremity surgery. WALANT does not require extensive medical clearance and allows for intraoperative assessment of range of motion. Additionally, it is frequently associated with lower costs and less postoperative pain when compared with traditional methods of anesthesia. Despite its expanded use for hand procedures, there are sparse data to support the use of WALANT in local and regional soft tissue flaps. Methods:. A retrospective review was performed. Twenty-one patients who underwent a local or regional soft tissue flap surgery using the WALANT technique from February 2, 2018 to February 25, 2022 were included in our analysis. Results:. Overall, one Louvre flap, two posterior tibial artery perforator propeller flaps, two reverse radial forearm flaps, two Quaba flaps, six cross finger flaps, one reverse homodigital island flap, three first dorsal metacarpal artery flaps, two thenar flaps, and two Moberg flaps were performed. Patients were followed up for an average of 11.9 ± 8.1 weeks. During this time, no postoperative complications occurred. All patients demonstrated appropriate healing at donor and recipient sites. Full range of motion was regained for all patients. Conclusions:. WALANT is safe and effective for use in local and regional soft tissue flap surgery. Surgeons should consider this technique for more involved procedures such as flap surgery, as preliminary results demonstrate positive outcomes and potentially superior recovery for patients

Loss of progesterone receptor-mediated actions induce preterm cellular and structural remodeling of the cervix and premature birth.

A decline in serum progesterone or antagonism of progesterone receptor function results in preterm labor and birth. Whether characteristics of premature remodeling of the cervix after antiprogestins or ovariectomy are similar to that at term was the focus of the present study. Groups of pregnant rats were treated with vehicle, a progesterone receptor antagonist (onapristone or mifepristone), or ovariectomized on day 17 postbreeding. As expected, controls given vehicle delivered at term while rats delivered preterm after progesterone receptor antagonist treatment or ovariectomy. Similar to the cervix before term, the preterm cervix of progesterone receptor antagonist-treated rats was characterized by reduced cell nuclei density, decreased collagen content and structure, as well as a greater presence of macrophages per unit area. Thus, loss of nuclear progesterone receptor-mediated actions promoted structural remodeling of the cervix, increased census of resident macrophages, and preterm birth much like that found in the cervix at term. In contrast to the progesterone receptor antagonist-induced advance in characteristics associated with remodeling, ovariectomy-induced loss of systemic progesterone did not affect hypertrophy, extracellular collagen, or macrophage numbers in the cervix. Thus, the structure and macrophage census in the cervix appear sufficient for premature ripening and birth to occur well before term. With progesterone receptors predominantly localized on cells other than macrophages, the findings suggest that interactions between cells may facilitate the loss of progesterone receptor-mediated actions as part of a final common mechanism that remodels the cervix in certain etiologies of preterm and with parturition at term

Photomicrographs of single (PR or ED1) and double-stained cells in sections of cervix from rats at term and with PR antagonist-induced preterm birth (RU486 PTB, day 18 postbreeding).

<p><b>A, B</b>. PR or ED1 stained section, Vector Red by alkaline phosphatase or brown HRP-DAB respectively, from a Con PP rat on day of term birth (day 22 postbreeding). <b>C, D</b>. PR- and ED1-stained cervix sections from a postpartum Con PP rat at term or RU486 PTB rat with preterm birth. Cell nuclei were visualized with a methyl green counterstain. These photomicrographs are representative of observations from analyses of cervix sections from 3–4 rats/group with available sections (Con D17.5, 18, PP; RU486 D17.5, PTB; Ona D17.5; Ovx D18. Double-stained PR and ED1 cells were a sparse minority of the total number of single PR or ED1 stained cells. Staining characteristics, including the predominance of single-stained cells, are consistent with a previous report in the cervix from postpartum women <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081340#pone.0081340-EkmanOrdeberg1" target="_blank">[32]</a>. Arrows indicate examples of single stained cells. Examples of putative PR+ED1-stained cells are highlighted in the box in panels B and D (magnified inset). Scale bars indicating 15 µm and 30 µm (inset), respectively. See Methods for details.</p

Degradation of collagen content and structure in the peripartum cervix at term and in preterm birth.

<p>Photomicrographs of picrosirius red dye-stained cervix sections from a rat that was <b>A</b>. nonpregnant (NP; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081340#pone.0081340-Boyd1" target="_blank">[30]</a>), or from rats in <b>B</b>. Con D21.5 preterm day 21.5 postbreeding, <b>C</b>. Ona PTB or <b>D</b>. Ovx PTB groups. Intensity of orange-red stain reflects extracellular collagen content and cross-linked fiber structure. Photomicrographs were obtained at magnifications of 40x, stitched, and sized to size. Scale bars indicate 1 mm. <b>E</b>. Optical density (mean±SE, n = 5–7 rats/group at each postbreeding date; analyses of 27 non-overlapping sections/rat) of picrosirius red-stained sections of cervix from Con, PR antagonist-treated, and Ovx rats normalized to cell nuclei number/area in each individual to account for tissue hypertrophy and edema. SE bars on some groups are small and obscured by the mean. Optical density is inversely related to collagen content and structure (details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081340#s2" target="_blank">Materials and Methods</a>). Group designations and statistical comparisons are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081340#pone-0081340-g001" target="_blank">Figure 1</a> legend. By example, the ^b assigned to the Ona D17.5 group indicates, by comparison, a significant decrease in OD of cervix sections from the RU486 D17.5, Ovx D17.5, and Ona PTB groups (greater collagen content and structure). By contrast, lack of a letter ^k indicates not significant differences in comparison to the Con D21.5 group. For perspective, the OD of cervix sections from all groups of pregnant rats were significantly increased, i.e., reduced collagen content and structure, compared to that in nonpregnant controls (1.506±0.0309 µm³ ×10⁻¹, n = 6 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081340#pone.0081340-Boyd1" target="_blank">[30]</a>; p<0.05 ANOVA).</p

Peripartum progesterone in circulation in preterm and term birth.

<p>Serum progesterone concentrations (mean±SE, n = 4–6/group) on various days postbreeding in controls, and groups of pregnant rats treated with Onapristone (Ona) or RU486 or bilaterally ovariectomized on the morning of day 17 of pregnancy (Ovx). Preterm birth (PTB) occurred on day 18 postbreeding for Ona- and RU486-treated rats, by 27 h or 26 h post-treatment, respectively (n = 5 and 6 respectively). PTB occurred by the morning of day 19 postbreeding in the Ovx group (36–44 h post-surgery, n = 5). Postpartum (PP) is the morning of the day of birth at term on day 22 postbreeding in Cons (n = 5). Typical estrous cycle serum progesterone concentrations range from 5–50 ng/ml in nonpregnant rats <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081340#pone.0081340-Smith1" target="_blank">[12]</a>. For statistical comparisons, groups were assigned an individual letter, consecutively from ^a for Con D17.5 to ^l for Con PP groups. The letter above a bar indicates p<0.05 vs the respective treatment group (ANOVA with Tukey's test for individual comparisons). By example, ^a over bars for Ona D17.5, RU486 D17.5 and Con PP groups indicates a significant difference for each versus the Con D17.5 group. The absence of a letter, ^k for example, indicates no statistical difference for any group compared to Con D21.5. See Methods for experimental details.</p

Photomicrographs of rat cervix sections for comparison of gross and cellular anatomy.

<p>Stitched multiple photomicrographs of a cervix section stained with the pan-macrophage marker ED-1 from <b>A</b>. a nonpregnant (NP, hematoxylin counterstain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081340#pone.0081340-Boyd1" target="_blank">[30]</a>) and <b>E</b>. a prepartum control rat (Con D21.5, day 21.5 postbreeding, methyl green counterstain). Green box highlights cervix lengthening and hypertrophy with pregnancy (scale bar = 1 mm). Vaginal folds surround the Os while uterine tissue body is generally above the green box, though a clear border between the cervix and uterus is difficult to define. Other photomicrographs of ED-1-stained, methyl green counterstained cervix sections from control rats on day 17.5 or 21.5 postbreeding or postpartum day 22 postbreeding (<b>B</b>. Con D17.5, <b>C</b>. Con D21.5, or <b>G</b>. Con PP, respectively) or from a rat treated with <b>D</b>. Ona or <b>H</b>. ovariectomized (Ovx) on day 17 postbreeding with preterm birth (PTB). <b>F</b>. Negative is a section from a Con D21.5 cervix that was processed without primary ED1 antibody (nonspecific background is not associated with methyl green-stained cell nucleus – example specified with black arrowhead). Box indicates area magnified in insets; examples of fully-stained macrophages with brown deposits are shown by arrows. Photomicrographs were taken at 40x and sized to fit; scale bars in 4 right panels are 25 μm and 5 μm for insets, respectively.</p