PLOS ONE: [sortOrder=DATE_NEWEST_FIRST, filterJournals=PLoSONE, sort=Date, newest first, q=subject:"Electrophoretic techniques"]PLOShttps://journals.plos.org/plosone/webmaster@plos.orgaccelerating the publication of peer-reviewed sciencehttps://journals.plos.org/plosone/search/feed/atom?sortOrder=DATE_NEWEST_FIRST&filterJournals=PLoSONE&sort=Date,+newest+first&unformattedQuery=subject:%22Electrophoretic+techniques%22All PLOS articles are Open Access.https://journals.plos.org/plosone/resource/img/favicon.icohttps://journals.plos.org/plosone/resource/img/favicon.ico2024-03-28T10:26:25ZPerformance evaluation of different albumin assays for the detection of analbuminemiaYi ZhangAfsoun AbdollahiChaylen AndolinoKeigo TomooBailey M. FosterUma K. AryalGregory C. Henderson10.1371/journal.pone.03001302024-03-06T14:00:00Z2024-03-06T14:00:00Z<p>by Yi Zhang, Afsoun Abdollahi, Chaylen Andolino, Keigo Tomoo, Bailey M. Foster, Uma K. Aryal, Gregory C. Henderson</p>
Analbuminemia is characterized by the near absence of albumin in the plasma. Different methods are available for measuring albumin levels, but they do not necessarily agree with one another. It is a concern that analbuminemic samples could be falsely characterized due to the incorrect estimation of albumin. The objective of the work was to evaluate the performance of different assays in detecting analbuminemia. Albumin knockout (Alb<sup>-/-</sup>) mouse plasma was used to test the suitability of different albumin assays for their ability to properly characterize extreme albumin deficiency. Bromocresol green (BCG), bromocresol purple (BCP), enzyme-linked immunosorbent assay (ELISA), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and gel electrophoresis were tested. The LC-MS/MS assay exhibited broad coverage of the amino acid sequence of albumin and indicated 8,400-fold lower (<i>P</i><0.0001) albumin expression in Alb<sup>-/-</sup> than wildtype (WT), demonstrating its suitability for identifying extreme albumin deficiency. ELISA estimated albumin at 1.5±0.1 g/dL in WT and was below the detection limit in all Alb<sup>-/-</sup> samples. Gel electrophoresis yielded consistent results with LC-MS/MS and ELISA. The BCG assay overestimated albumin with apparently appreciable albumin concentrations in Alb<sup>-/-</sup> mice, yet the assay still indicated a significant difference between genotypes (Alb<sup>-/-</sup>, 1.2±0.05 g/dL, WT, 3.7±0.1 g/dL, <i>P</i><0.0001). BCP drastically overestimated albumin and could not successfully identify the known analbuminemic phenotype of Alb<sup>-/-</sup> mice. By using Alb<sup>-/-</sup> plasma as a reference material and LC-MS/MS as a reference method, ELISA and gel electrophoresis appear appropriate for identifying analbuminemia, while BCG and BCP are not suitable. It is concluded that dye-binding assays should be avoided when extreme hypoalbuminemia or analbuminemia is suspected.Rapid and specific detection of wheat spindle streak mosaic virus using RT-LAMP in durum wheat crude leaf extractMonica MarraPaolo MussanoEugenio PintonCinzia MontemurroElena BaldoniClaudio RattiSlavica MatićChiara D’ErricoGian Paolo Accotto10.1371/journal.pone.02990782024-02-29T14:00:00Z2024-02-29T14:00:00Z<p>by Monica Marra, Paolo Mussano, Eugenio Pinton, Cinzia Montemurro, Elena Baldoni, Claudio Ratti, Slavica Matić, Chiara D’Errico, Gian Paolo Accotto</p>
To accurately determine the spread of any pathogen, including plant viruses, a quick, sensitive, cost-effective, point-of-care diagnostic assay is necessary. Wheat spindle streak mosaic virus (WSSMV) is a <i>Bymovirus</i>, transmitted by the plasmodiophorid <i>Polymyxa graminis</i> Led, which causes yellow mosaic and reduces the grain yield in wheat. Currently, detection protocols for WSSMV use ELISA or more sensitive PCR-based approaches requiring specialized laboratory and personnel. A protocol for reverse transcription loop mediated isothermal amplification (RT-LAMP) has been developed and optimized for the rapid detection of viruses using crude extracts from wheat leaves. The protocol was specific for WSSMV detection, while no reaction was observed with SBCMV or SBWMV, the non-target viruses transmitted by the same vector. The RT-LAMP assay was shown to be as sensitive as the one-step WSSMV specific RT-PCR. The RT-LAMP assay can be performed under field conditions using a portable instrument, and can help the actual spread of WSSMV, an aspect of this virus not yet well understood, to be explored.Extraction and selection of high-molecular-weight DNA for long-read sequencing from <i>Chlamydomonas reinhardtii</i>Frédéric ChauxNicolas AgierStephan EberhardZhou Xu10.1371/journal.pone.02970142024-02-08T14:00:00Z2024-02-08T14:00:00Z<p>by Frédéric Chaux, Nicolas Agier, Stephan Eberhard, Zhou Xu</p>
Recent advances in long-read sequencing technologies have enabled the complete assembly of eukaryotic genomes from telomere to telomere by allowing repeated regions to be fully sequenced and assembled, thus filling the gaps left by previous short-read sequencing methods. Furthermore, long-read sequencing can also help characterizing structural variants, with applications in the fields of genome evolution or cancer genomics. For many organisms, the main bottleneck to sequence long reads remains the lack of robust methods to obtain high-molecular-weight (HMW) DNA. For this purpose, we developed an optimized protocol to extract DNA suitable for long-read sequencing from the unicellular green alga <i>Chlamydomonas reinhardtii</i>, based on CTAB/phenol extraction followed by a size selection step for long DNA molecules. We provide validation results for the extraction protocol, as well as statistics obtained with Oxford Nanopore Technologies sequencing.Development of modified CTAB and Trizol protocols to isolate high molecular weight (HMW) RNA from polyphenol and polysaccharides rich pigeonpea (<i>Cajanuscajan</i> (L.) MillspPawan MainkarDeepanshu JayaswalDeepesh KumarKuldip JayaswallSandeep JaiswalArvind Nath SinghSanjay KumarRekha Kansal10.1371/journal.pone.02919492023-12-08T14:00:00Z2023-12-08T14:00:00Z<p>by Pawan Mainkar, Deepanshu Jayaswal, Deepesh Kumar, Kuldip Jayaswall, Sandeep Jaiswal, Arvind Nath Singh, Sanjay Kumar, Rekha Kansal</p>
Pigeonpea (<i>Cajanuscajan</i> L.) is a legume crop that contains high levels of polyphenolic compounds and polysaccharides that become a hindrance in extracting good-quality and enough amount of RNA from its tissues. With the existing methods of RNA isolation, the phenolic compounds may co-precipitate or bind to the RNA giving false results. Therefore, in the present study, we have modified conventional CTAB and Trizol-based methods which resulted in good quality with the absorbance A260/A280 ratios in the range of 1.83 to 1.98 and A260/230 ratios in the range of 2.0–2.23, revealed RNA to be of high purity and free of contaminants. Both of the proposed protocols yielded a good quantity of RNA ranging from 289 to 422μg per gram of tissue. Distinctly visible bands of 28S and 18S rRNA were observed without degradation or smear, which indicated the presence of intact RNA. RT-PCR analysis showed that isolated RNA was quantitatively sufficient and compliant for the subsequent gene expression analysis.The telomere resolvase, TelA, utilizes an underwound pre-cleavage intermediate to promote hairpin telomere formationMahrokh BalouchiShu Hui HuangSiobhan L. McGrathKerri Kobryn10.1371/journal.pone.02947322023-11-29T14:00:00Z2023-11-29T14:00:00Z<p>by Mahrokh Balouchi, Shu Hui Huang, Siobhan L. McGrath, Kerri Kobryn</p>
The telomere resolvase, TelA, forms the hairpin telomeres of the linear chromosome of <i>Agrobacterium tumefaciens</i> in a process referred to as telomere resolution. Telomere resolution is a unique DNA cleavage and rejoining reaction that resolves replicated telomere junctions into a pair of hairpin telomeres. Telomere resolvases utilize a reaction mechanism with similarities to that of topoisomerase-IB enzymes and tyrosine recombinases. The reaction proceeds without the need for high-energy cofactors due to the use of a covalent, enzyme-cleaved DNA intermediate that stores the bond energy of the cleaved bonds in 3’-phosphotyrosyl linkages. The cleaved DNA strands are then refolded into a hairpin conformation and the 5’-OH ends of the refolded strands attack the 3’-phosphotyrosine linkages in order to rejoin the DNA strands into hairpin telomeres. Because this kind of reaction mechanism is, in principle, reversible it is unclear how TelA controls the direction of the reaction and propels the reaction to completion. We present evidence that TelA forms and/or stabilizes a pre-cleavage intermediate that features breakage of the four central basepairs between the scissile phosphates prior to DNA cleavage to help propel the reaction forwards, thus preventing abortive cleavage and rejoining cycles that regenerate the substrate DNA. We identify eight TelA sidechains, located in the hairpin-binding module and catalytic domains of TelA, implicated in this process. These mutants were deficient for telomere resolution on parental replicated telomere junctions but were rescued by introduction of substrate modifications that mimic unwinding of the DNA between the scissile phosphates.Automation protocol for high-efficiency and high-quality genomic DNA extraction from <i>Saccharomyces cerevisiae</i>Nina AlperovichBenjamin M. ScottDavid Ross10.1371/journal.pone.02924012023-10-17T14:00:00Z2023-10-17T14:00:00Z<p>by Nina Alperovich, Benjamin M. Scott, David Ross</p>
Although many protocols have been previously developed for genomic DNA (gDNA) extraction from <i>S</i>. <i>cerevisiae</i>, to take advantage of recent advances in laboratory automation and DNA-barcode sequencing, there is a need for automated methods that can provide high-quality gDNA at high efficiency. Here, we describe and demonstrate a fully automated protocol that includes five basic steps: cell wall and RNA digestion, cell lysis, DNA binding to magnetic beads, washing with ethanol, and elution. Our protocol avoids the use of hazardous reagents (e.g., phenol, chloroform), glass beads for mechanical cell disruption, or incubation of samples at 100°C (i.e., boiling). We show that our protocol can extract gDNA with high efficiency both from cells grown in liquid culture and from colonies grown on agar plates. We also show results from gel electrophoresis that demonstrate that the resulting gDNA is of high quality.Are rapid tests and confirmatory western blot used for cattle and small ruminants TSEs reliable tools for the diagnosis of Chronic Wasting Disease in Europe?Maria MazzaLinh TranDaniela LopreviteMaria C. CavarrettaDaniela MeloniLuana Dell’AttiJørn VågeKnut MadslienTram T. VuongElena BozzettaSylvie L. Benestad10.1371/journal.pone.02862662023-08-30T14:00:00Z2023-08-30T14:00:00Z<p>by Maria Mazza, Linh Tran, Daniela Loprevite, Maria C. Cavarretta, Daniela Meloni, Luana Dell’Atti, Jørn Våge, Knut Madslien, Tram T. Vuong, Elena Bozzetta, Sylvie L. Benestad</p>
The first case of CWD in Europe was detected in a Norwegian reindeer in 2016, followed later by two CWD cases in Norwegian moose. To prevent the potential spread of CWD to the EU, the European Commission (Regulation EU 2017_1972) implemented a CWD surveillance programme in cervids in the six countries having reindeer and or moose (Estonia, Finland, Latvia, Lithuania, Poland, and Sweden). Each country had to test a minimum of 3000 cervids for CWD using diagnostic rapid tests approved by the EC Regulation. Experimental transmission studies in rodents have demonstrated that the CWD strains found in Norwegian reindeer are different from those found in moose and that these European strains are all different from the North American ones. Data on the performances of authorised rapid tests are limited for CWD (from North America) and are currently minimal for CWD from Europe, due to the paucity of positive material. The aim of this study was to evaluate the diagnostic performances of three of the so-called “rapid” tests, commercially available and approved for TSE diagnosis in cattle and small ruminants, to detect the CWD strains circulating in Europe. The performances of these three tests were also compared to two different confirmatory western blot methods. Using parallel testing on the same panel of available samples, we evaluated here the analytical sensitivity of these methods for TSE diagnosis of CWD in Norwegian cervids tissues. Our results show that all the methods applied were able to detect the CWD positive samples even if differences in analytical sensitivity were clearly observed. Although this study could not assess the test accuracy, due to the small number of samples available, it is conceivable that the rapid and confirmatory diagnostic systems applied for CWD surveillance in Northern Europe are reliable tools.Purification and characterization of novel isoforms of the polyphenol oxidase from <i>Malus domestica</i> fruit pulpNaila SajjadM. Sheeraz AhmadRaja Tahir MahmoodMuhammad TariqMuhammad Javaid AsadShamaila IrumAnisa AndleebAbid RiazDawood Ahmed10.1371/journal.pone.02760412023-08-25T14:00:00Z2023-08-25T14:00:00Z<p>by Naila Sajjad, M. Sheeraz Ahmad, Raja Tahir Mahmood, Muhammad Tariq, Muhammad Javaid Asad, Shamaila Irum, Anisa Andleeb, Abid Riaz, Dawood Ahmed</p>
Polyphenol oxidases (PPOs), belong to the group of oxidoreductases that are copper containing enzymes and are responsible for plant browning. PPOs are extensively distributed in plant kingdom and can oxidize wide range of aromatic compounds of industrial importance. The aim of this study was purification and characterization of PPO isoforms from the fruit pulp of Golden delicious apple. High performance liquid chromatography was used to purify the two novel isoforms of PPO and further their molecular weights (45 and 28 kDa) were determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified isoforms have optimum pH (6.5), optimum temperature (40°C), the V<sub>max</sub> (4.45 μM/min) and K<sub>m</sub> (74.21 mM) with catechol substrate. The N-terminal microsequences of both PPO isoforms were determined using a pulse liquid protein sequencer and found to be AKITFHG (28 kDa) and APGGG (45 kDa). Polyphenol oxidases are efficiently used in the pharmaceutical, paper and pulp, textiles and food industries. Recently, the PPOs have been used for bioremediation and in the development of biosensors.Synthesis of length-tunable DNA carriers for nanopore sensingZachary RoelenVincent Tabard-Cossa10.1371/journal.pone.02905592023-08-23T14:00:00Z2023-08-23T14:00:00Z<p>by Zachary Roelen, Vincent Tabard-Cossa</p>
Molecular carriers represent an increasingly common strategy in the field of nanopore sensing to use secondary molecules to selectively report on the presence of target analytes in solution, allowing for sensitive assays of otherwise hard-to-detect molecules such as small, weakly-charged proteins. However, existing carrier designs can often introduce drawbacks to nanopore experiments including higher levels of cost/complexity and carrier-pore interactions that lead to ambiguous signals and elevated clogging rates. In this work, we present a simple method of carrier production based on sticky-ended DNA molecules that emphasizes ease-of-synthesis and compatibility with nanopore sensing and analysis. In particular, our method incorporates the ability to flexibly control the length of the DNA carriers produced, enhancing the multiplexing potential of this carrier system through the separable nanopore signals they could generate for distinct targets. A proof-of-concept nanopore experiment is also presented, involving carriers produced by our method with multiple lengths and attached to DNA nanostructure targets, in order to validate the capabilities of the system. As the breadth of applications for nanopore sensors continues to expand, the availability of tools such as those presented here to help translate the outcomes of these applications into robust nanopore signals will be of major importance.Tau protein aggregation associated with SARS-CoV-2 main proteaseRaphael Josef EberleMônika Aparecida CoronadoIan GeringSimon SommerhageKarolina KorostovAnja StefanskiKai StühlerVictoria Kraemer-SchulienLara BlömekeOliver BannachDieter Willbold10.1371/journal.pone.02881382023-08-21T14:00:00Z2023-08-21T14:00:00Z<p>by Raphael Josef Eberle, Mônika Aparecida Coronado, Ian Gering, Simon Sommerhage, Karolina Korostov, Anja Stefanski, Kai Stühler, Victoria Kraemer-Schulien, Lara Blömeke, Oliver Bannach, Dieter Willbold</p>
The primary function of virus proteases is the proteolytic processing of the viral polyprotein. These enzymes can also cleave host cell proteins, which is important for viral pathogenicity, modulation of cellular processes, viral replication, the defeat of antiviral responses and modulation of the immune response. It is known that COVID-19 can influence multiple tissues or organs and that infection can damage the functionality of the brain in multiple ways. After COVID-19 infections, amyloid-β, neurogranin, tau and phosphorylated tau were detected extracellularly, implicating possible neurodegenerative processes. The present study describes the possible induction of tau aggregation by the SARS-CoV-2 3CL protease (3CL<sup>pro</sup>) possibly relevant in neuropathology. Further investigations demonstrated that tau was proteolytically cleaved by the viral protease 3CL and, consequently, generated aggregates. However, more evidence is needed to confirm that COVID-19 is able to trigger neurodegenerative diseases.Key pathological features characterize minimal change disease-like IgA nephropathyTsung-Yueh WangFu-Pang ChangAn-Hang YangShuk-Man KaAnn ChenJyh-Tong HsiehFan-Yu ChenTsung-Lun LeePo-Yu TsengMing-Tsun TsaiSzu-Yuan LiChih-Yu YangJinn-Yang ChenChih-Ching LinDer-Cherng Tarng10.1371/journal.pone.02883842023-07-20T14:00:00Z2023-07-20T14:00:00Z<p>by Tsung-Yueh Wang, Fu-Pang Chang, An-Hang Yang, Shuk-Man Ka, Ann Chen, Jyh-Tong Hsieh, Fan-Yu Chen, Tsung-Lun Lee, Po-Yu Tseng, Ming-Tsun Tsai, Szu-Yuan Li, Chih-Yu Yang, Jinn-Yang Chen, Chih-Ching Lin, Der-Cherng Tarng</p>
Aims <p>A subset of IgA nephropathy (IgAN) patients exhibiting minimal change disease (MCD) like features present with nephrotic-range proteinuria and warrants immunosuppressive therapy (IST). However, the diagnosis of MCD-like IgAN varied by reports. We aimed to identify the key pathological features of MCD-like IgAN.</p> Methods <p>In this cohort, 228 patients had biopsy-proven IgAN from 2009 to 2021, of which 44 without segmental sclerosis were enrolled. Patients were classified into segmental (< 50% glomerular capillary loop involvement) or global (> 50%) foot process effacement (FPE) groups. We further stratified them according to the usage of immunosuppressant therapy after biopsy. Clinical manifestations, treatment response, and renal outcome were compared.</p> Results <p>26 cases (59.1%) were classified as segmental FPE group and 18 cases (40.9%) as global FPE group. The global FPE group had more severe proteinuria (11.48 [2.60, 15.29] vs. 0.97 [0.14, 1.67] g/g, <i>p</i> = 0.001) and had a higher proportion of complete remission (81.8% vs. 20%, <i>p</i> = 0.018). In the global FPE group, patients without IST experienced more rapid downward eGFR change than the IST-treated population (-0.38 [-1.24, 0.06] vs. 1.26 [-0.17, 3.20]mL/min/1.73 m<sup>2</sup>/month, <i>p</i> = 0.004).</p> Conclusions <p>The absence of segmental sclerosis and the presence of global FPE are valuable pathological features that assist in identifying MCD-like IgAN.</p>FAVIS: Fast and versatile protocol for non-destructive metabarcoding of bulk insect samplesElzbieta Iwaszkiewicz-EggebrechtPiotr ŁukasikMateusz BuczekJunchen DengEmily A. HartopHarald HavnåsMonika Prus-FrankowskaCarina R. UgarphPaulina ViteriAnders F. AnderssonTomas RoslinAyco J. M. TackFredrik RonquistAndreia Miraldo10.1371/journal.pone.02862722023-07-19T14:00:00Z2023-07-19T14:00:00Z<p>by Elzbieta Iwaszkiewicz-Eggebrecht, Piotr Łukasik, Mateusz Buczek, Junchen Deng, Emily A. Hartop, Harald Havnås, Monika Prus-Frankowska, Carina R. Ugarph, Paulina Viteri, Anders F. Andersson, Tomas Roslin, Ayco J. M. Tack, Fredrik Ronquist, Andreia Miraldo</p>
Insects are diverse and sustain essential ecosystem functions, yet remain understudied. Recent reports about declines in insect abundance and diversity have highlighted a pressing need for comprehensive large-scale monitoring. Metabarcoding (high-throughput bulk sequencing of marker gene amplicons) offers a cost-effective and relatively fast method for characterizing insect community samples. However, the methodology applied varies greatly among studies, thus complicating the design of large-scale and repeatable monitoring schemes. Here we describe a non-destructive metabarcoding protocol that is optimized for high-throughput processing of Malaise trap samples and other bulk insect samples. The protocol details the process from obtaining bulk samples up to submitting libraries for sequencing. It is divided into four sections: 1) Laboratory workspace preparation; 2) Sample processing—decanting ethanol, measuring the wet-weight biomass and the concentration of the preservative ethanol, performing non-destructive lysis and preserving the insect material for future work; 3) DNA extraction and purification; and 4) Library preparation and sequencing. The protocol relies on readily available reagents and materials. For steps that require expensive infrastructure, such as the DNA purification robots, we suggest alternative low-cost solutions. The use of this protocol yields a comprehensive assessment of the number of species present in a given sample, their relative read abundances and the overall insect biomass. To date, we have successfully applied the protocol to more than 7000 Malaise trap samples obtained from Sweden and Madagascar. We demonstrate the data yield from the protocol using a small subset of these samples.Towards a barrier-free anthropomorphic brain phantom for quantitative magnetic resonance imaging: Design, first construction attempt, and challengesMikail KraftSlavka RygerBen P. BermanMatthew E. DownsKalina V. JordanovaMegan E. PoormanSamuel D. OberdickStephen E. OgierStephen E. RussekJoseph DagherKathryn E. Keenan10.1371/journal.pone.02854322023-07-12T14:00:00Z2023-07-12T14:00:00Z<p>by Mikail Kraft, Slavka Ryger, Ben P. Berman, Matthew E. Downs, Kalina V. Jordanova, Megan E. Poorman, Samuel D. Oberdick, Stephen E. Ogier, Stephen E. Russek, Joseph Dagher, Kathryn E. Keenan</p>
Existing magnetic resonance imaging (MRI) reference objects, or phantoms, are typically constructed from simple liquid or gel solutions in containers with specific geometric configurations to enable multi-year stability. However, there is a need for phantoms that better mimic the human anatomy without barriers between the tissues. Barriers result in regions without MRI signal between the different tissue mimics, which is an artificial image artifact. We created an anatomically representative 3D structure of the brain that mimicked the T1 and T2 relaxation properties of white and gray matter at 3 T. While the goal was to avoid barriers between tissues, the 3D printed barrier between white and gray matter and other flaws in the construction were visible at 3 T. Stability measurements were made using a portable MRI system operating at 64 mT, and T2 relaxation time was stable from 0 to 22 weeks. The phantom T1 relaxation properties did change from 0 to 10 weeks; however, they did not substantially change between 10 weeks and 22 weeks. The anthropomorphic phantom used a dissolvable mold construction method to better mimic anatomy, which worked in small test objects. The construction process, though, had many challenges. We share this work with the hope that the community can build on our experience.Identification and initial characterization of Hfq-associated sRNAs in <i>Histophilus somni</i> strain 2336Bindu SubhadraDianjun CaoRoderick JensenClayton CaswellThomas J. Inzana10.1371/journal.pone.02861582023-05-23T14:00:00Z2023-05-23T14:00:00Z<p>by Bindu Subhadra, Dianjun Cao, Roderick Jensen, Clayton Caswell, Thomas J. Inzana</p>
Small RNAs (sRNA), in association with the global chaperone regulator Hfq, positively or negatively regulate gene expression in bacteria. For this study, <i>Histophilus somni</i> sRNAs that bind to Hfq were identified and then partially characterized. The Hfq-associated sRNAs in <i>H</i>. <i>somni</i> were isolated and identified by co-immunoprecipitation using anti-Hfq antibody, followed by sRNA sequencing. Sequence analysis of the sRNA samples identified 100 putative sRNAs, out of which 16 were present in pathogenic strain 2336, but not in non-pathogenic strain 129Pt. Bioinformatic analyses suggested that the sRNAs HS9, HS79, and HS97 could bind to many genes putatively involved in virulence/biofilm formation. Furthermore, multi-sequence alignment of the sRNA regions in the genome revealed that HS9 and HS97 could interact with sigma 54, which is a transcription factor linked to important bacterial traits, including motility, virulence, and biofilm formation. Northern blotting was used to determine the approximate size, abundance and any processing events attributed to the sRNAs. Selected sRNA candidates were confirmed to bind Hfq, as determined by electrophoretic mobility shift assays using sRNAs synthesized by <i>in vitro</i> transcription and recombinant Hfq. The exact transcriptional start site of the sRNA candidates was determined by RNA ligase-mediated rapid amplification of cDNA ends, followed by cloning and sequencing. This is the first investigation of <i>H</i>. <i>somni</i> sRNAs that show they may have important regulatory roles in virulence and biofilm formation.Structural requirements of <i>Holothuria floridana</i> fucosylated chondroitin sulfate oligosaccharides in anti-SARS-CoV-2 and anticoagulant activitiesMarwa FarragRohini DwivediPoonam SharmaDeepak KumarRitesh TandonVitor H. Pomin10.1371/journal.pone.02855392023-05-11T14:00:00Z2023-05-11T14:00:00Z<p>by Marwa Farrag, Rohini Dwivedi, Poonam Sharma, Deepak Kumar, Ritesh Tandon, Vitor H. Pomin</p>
Fucosylated chondroitin sulfate (FucCS) is a unique glycosaminoglycan found primarily in sea cucumbers. This marine sulfated glycan is composed of a chondroitin sulfate backbone decorated with fucosyl branches attached to the glucuronic acid. FucCS exhibits potential biological actions including inhibition of blood clotting and severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection. These biological effects have been attributed to certain structural features, including molecular weight (MW), and/or those related to fucosylation, such as degrees of fucosyl branches, sulfation patterns and contents. In a previous work, we were able to generate oligosaccharides of the FucCS from <i>Pentacta pygmaea</i> (PpFucCS) with reduced anticoagulant effect but still retaining significant anti-SARS-CoV-2 activity against the delta strain. In this work, we extended our study to the FucCS extracted from the species <i>Holothuria floridana</i> (HfFucCS). The oligosaccharides were prepared by free-radical depolymerization of the HfFucCS via copper-based Fenton reaction. One-dimensional <sup>1</sup>H nuclear magnetic resonance spectra were employed in structural analysis. Activated partial thromboplastin time and assays using protease (factors Xa and IIa) and serine protease inhibitors (antithrombin, and heparin cofactor II) in the presence of the sulfated carbohydrates were used to monitor anticoagulation. Anti-SARS-CoV-2 effects were measured using the concentration–response inhibitory curves of HEK-293T-human angiotensin-converting enzyme-2 cells infected with a baculovirus pseudotyped SARS-CoV-2 wild-type and delta variant spike (S)-proteins. Furthermore, the cytotoxicity of native HfFucCS and its oligosaccharides was also assessed. Like for PpFucCS, we were able to generate a HfFucCS oligosaccharide fraction devoid of high anticoagulant effect but still retaining considerable anti-SARS-CoV-2 actions against both variants. However, compared to the oligosaccharide fraction derived from PpFucCS, the average MW of the shortest active HfFucCS oligosaccharide fraction was significantly lower. This finding suggests that the specific structural feature in HfFucCS, the branching 3,4-di-sulfated fucoses together with the backbone 4,6-di-sulfated N-acetylgalactosamines, is relevant for the anti-SARS-CoV-2 activity of FucCS molecules.