Small Subunit Research Articles

Ribonucleotide reductase small subunit M2 promotes the proliferation of esophageal squamous cell carcinoma cells via HuR-mediated mRNA stabilization

Esophageal squamous cell carcinoma (ESCC), a malignancy of the digestive system, is highly prevalent and the primary cause of cancer-related deaths worldwide due to the lack of early diagnostic biomarkers and effective therapeutic targets. Dysregulated ribonucleotide reductase (RNR) expression has been confirmed to be causally linked to tumorigenesis. This study demonstrated that ribonucleotide reductase small subunit M2 (RRM2) is significantly upregulated in ESCC tissue and that its expression is negatively correlated with clinical outcomes. Mechanistically, HuR promotes RRM2 mRNA stabilization by binding to the adenine/uridine (AU)-rich elements (AREs) within the 3′UTR, resulting in persistent overexpression of RRM2. Furthermore, bifonazole is identified as an inhibitor of HuR via computational screening and molecular docking analysis. Bifonazole disrupts HuR-ARE interactions by competitively binding to HuR at F65, R97, I103, and R153 residues, resulting in reduced RRM2 expression. Furthermore, bifonazole exhibited antitumor effects on ESCC patient-derived xenograft (PDX) models by decreasing RRM2 expression and the dNTP pool. In summary, this study reveals the interaction network among HuR, RRM2, and bifonazole and demonstrated that bifonazole is a potential therapeutic compound for ESCC through inhibition of the HuR/RRM2 axis.

Evaluation of RuBisCO gene expression in Caragana arborescens f. pyramidalis LAM. under drought conditions

Abstract At present, there is an increase in the concentration of CO2 in the atmosphere, which leads to significant climatic changes, especially in arid areas. Tree and shrub species provide fixation of atmospheric carbon dioxide, ensuring assimilation of inorganic carbon used in photosynthesis, as well as reducing the greenhouse effect. The enzyme Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) binds CO2 and triggers the Calvin cycle. The aim of the study was to evaluate the expression of the large subunit of the RuBisCO gene (RbcL) in Caragana arborescens f. pyramidalis LAM. and the in-house selected variety Caragana arborescens f. pyramidalis LAM. “Incomparable VNIALMI”, growing under drought conditions on the Kirov forestry (North of the Ergeninskaya Upland) with a characteristic arid climate. RNA was isolated from leaf blades collected at the end of fruiting. RbcL expression was evaluated by RT-qPCR using a universal primer pair selected by us. The study determined that the vital state in C. arborescens f. pyramidalis and C. arborescens f. pyramidalis ‘Incomparable VNIALMI’ was rated as good and that they were adapted to arid climate. Other analysis of RbcL expression showed lower values of amplification threshold cycles in C. arborescens f. pyramidalis LAM. “Incomparable VNIALMI”.

Unveiling the mechanism of hesperidin-induced LdTopI-mediated cell death pathway in protozoan parasite Leishmania donovani

Unicellular protozoan parasite Leishmania donovani is the causative agent for visceral leishmaniasis (VL) or Kala-azar, a neglected fatal parasitic disease. The conventional treatment of VL consists of therapeutic agents having several shortcomings such as toxicity, high cost, efficacy variance and increased drug resistance. Therefore, there is a desperate need to develop an effective treatment against the parasite. Previous reports suggested that flavonoids can inhibit the enzyme Leishmania donovani DNA topoisomerase I (LdTopILS). Therefore, for the first time in this present study, we divulge HSP (one of the natural sources of flavonoids), as a potent natural antileishmanial compound with efficacy in BALB/c mice at 20 mg/kg of body weight, inhibits LdTopILS at 97 % of its activity at 160 μM in preincubation condition (competitively). It binds with free enzyme and does not allow it to bind with the substrate DNA. Moreover, HSP does not stabilize DNA-topoisomerase I cleavable complex. Thus, HSP acts a catalytic topoisomerase I inhibitor, which inhibits complete activity by binding with Lys269 and Thr411 of large subunit of the enzyme. On the other hand, HSP induces the topo I-mediated programmed cell death process by the formation of cellular reactive oxygen species, resulting in depolarization of mitochondrial membrane potential, followed by fragmentation of nuclear DNA. Therefore, the present study illuminates a natural flavonoid that in future might be a promising lead for the treatment of VL.

The chloroplast pentatricopeptide repeat protein RCN22 regulates tiller number in rice by affecting sugar levels via the TB1–RCN22–RbcL module

As an important yield component, rice tiller number controls panicle number and determines grain yield. Regulation of rice tiller number by chloroplast pentatricopeptide repeat (PPR) proteins has not been reported previously. Here, we report the rice reduced culm number22 (rcn22) mutant, which produces few tillers owing to suppressed tiller bud elongation. Map-based cloning revealed that RCN22 encodes a chloroplast-localized P-type PPR protein. We found that RCN22 specifically binds to the 5′ UTR of RbcL mRNA (encoding the large subunit of Rubisco) and enhances its stability. The reduced abundance of RbcL mRNA in rcn22 leads to a lower photosynthetic rate and decreased sugar levels. Consequently, transcript levels of DWARF3 (D3) and TEOSINTE BRANCHED1 (TB1) (which encode negative regulators of tiller bud elongation) are increased, whereas protein levels of the positive regulator DWARF53 (D53) are decreased. Furthermore, high concentrations of sucrose can rescue the tiller bud growth defect of the rcn22 mutant. On the other hand, TB1 directly binds to the RCN22 promoter and downregulates its expression. The tb1/rcn22 double mutant shows a tillering phenotype similar to that of rcn22. Our results suggest that the TB1–RCN22–RbcL module plays a vital role in rice tiller bud elongation by affecting sugar levels.

Cyberlindnera qingyuanensis f.a., sp. nov., a yeast species isolated from rotting wood.

Two yeast strains were isolated from rotting wood samples collected on Qingyuan Mountain, Fujian Province, PR China. Phylogenetic analysis, based on the concatenated sequences of the internal transcribed spacer (ITS) region and the D1/D2 domain of the large subunit rRNA gene, revealed that these two strains represent a novel species of the genus Cyberlindnera. The proposed name for this new species is Cyberlindnera qingyuanensis f.a., sp. nov. (holotype: GDMCC 2.300; ex-type: PYCC 9925) although the formation of ascospores was not observed. The novel species differs from its close relative Cyberlindnera galapagoensis by 7.7% sequence divergence (37 substitutions and seven indels) in the D1/D2 domain and 9.7% sequence divergence (42 substitutions and 34 indels) in the ITS region, respectively. Additionally, Cyb. qingyuanensis differs from its close relative Cyb. galapagoensis by its ability to grow in cellobiose, l-rhamnose, ribitol, galactitol, and dl-lactate, its growth at 37 °C, and its inability to ferment raffinose. The Mycobank number is MB 854693.

Protective RBD-dimer vaccines against SARS-CoV-2 and its variants produced in glycoengineered Pichia pastoris.

Protective vaccines are crucial for preventing and controlling coronavirus disease 2019 (COVID-19). Updated vaccines are needed to confront the continuously evolving and circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. These vaccines should be safe, effective, amenable to easily scalable production, and affordable. Previously, we developed receptor binding domain (RBD) dimer-based protein subunit vaccines (ZF2001 and updated vaccines) in mammalian cells. In this study, we explored a strategy for producing RBD-dimer immunogens in Pichia pastoris. We found that wild-type P. pastoris produced hyperglycosylated RBD-dimer protein containing four N-glycosylation sites in P. pastoris. Therefore, we engineered the wild type P. pastoris (GS strain) into GSΔOCH1pAO by deleting the OCH1 gene (encoding α-1,6-mannosyltransferase enzyme) to decrease glycosylation, as well as by overexpressing the HIS4 gene (encoding histidine dehydrogenase) to increase histidine synthesis for better growth. In addition, RBD-dimer protein was truncated to remove the R328/F329 cleavage sites in P. pastoris. Several homogeneous RBD-dimer proteins were produced in the GSΔOCH1pAO strain, demonstrating the feasibility of using the P. pastoris expression system. We further resolved the cryo-EM structure of prototype-Beta RBD-dimer complexed with the neutralizing antibody CB6 to reveal the completely exposed immune epitopes of the RBDs. In a murine model, we demonstrated that the yeast-produced RBD-dimer induces robust and protective antibody responses, which is suitable for boosting immunization. This study developed the yeast system for producing SARS-CoV-2 RBD-dimer immunogens, providing a promising platform and pipeline for the future continuous updating and production of SARS-CoV-2 vaccines.

Heterologous Prime-Boost Immunization Strategies Using Varicella-Zoster Virus gE mRNA Vaccine and Adjuvanted Protein Subunit Vaccine Triggered Superior Cell Immune Response in Middle-Aged Mice.

Heterologous immunization using different vaccine platforms has been demonstrated as an efficient strategy to enhance antigen-specific immune responses. In this study, we performed a head-to-head comparison of both humoral and cellular immune response induced by different prime-boost immunization regimens of mRNA vaccine and adjuvanted protein subunit vaccine against varicella-zoster virus (VZV) in middle-aged mice, aiming to get a better understanding of the influence of vaccination schedule on immune response. VZV glycoprotein (gE) mRNA was synthesized and encapsulated into SM-102-based lipid nanoparticles (LNPs). VZV-primed middle-aged C57BL/6 mice were then subjected to homologous and heterologous prime-boost immunization strategies using VZV gE mRNA vaccine (RNA-gE) and protein subunit vaccine (PS-gE). The antigen-specific antibodies were evaluated using enzyme-linked immunosorbent assay (ELISA) analysis. Additionally, cell-mediated immunity (CMI) was detected using ELISPOT assay and flow cytometry. Besides, in vivo safety profiles were also evaluated and compared. The mRNA-loaded lipid nanoparticles had a hydrodynamic diameter of approximately 130 nm and a polydispersity index of 0.156. Total IgG antibody levels exhibited no significant differences among different immunization strategies. However, mice received 2×RNA-gE or RNA-gE>PS-gE showed a lower IgG1/IgG2c ratio than those received 2×PS-gE and PS-gE> RNA-gE. The CMI response induced by 2×RNA-gE or RNA-gE>PS-gE was significantly stronger than that induced by 2×PS-gE and PS-gE> RNA-gE. The safety evaluation indicated that both mRNA vaccine and protein vaccine induced a transient body weight loss in mice. Furthermore, the protein vaccine produced a notable inflammatory response at the injection sites, while the mRNA vaccine showed no observable inflammation. The heterologous prime-boost strategy has demonstrated that an mRNA-primed immunization regimen can induce a better cell-mediated immune response than a protein subunit-primed regimen in middle-aged mice. These findings provide valuable insights into the design and optimization of VZV vaccines with the potentials to broaden varicella vaccination strategies in the future.

Recent research advances in the development of Dabie Banda virus vaccines.

Severe fever with thrombocytopenia syndrome (SFTS) is a newly identified tick-borne viral hemorrhagic fever caused by Dabie Banda virus (DBV). The virus was first discovered in eastern China in 2009 and is now considered an infectious disease with a mortality rate ranging from 6.3% to 30%. The best strategy for controlling SFTS is to develop effective vaccines. However, no approved vaccines are currently available to prevent this disease, despite the number of extensive and in-depth studies conducted on DBV in the past few years. This review focuses on the structure of DBV and the induced host immune responses which are the fundamental factors in vaccine development, and thoroughly summarizes the current research progress on DBV vaccines. The developing DBV vaccines include protein subunit vaccines, live attenuated vaccines, recombinant virus vector vaccines, and DNA vaccines. At present, almost all candidate vaccines for DBV are in the laboratory development or preclinical stages. There remain challenges in successfully developing clinically approved DBV vaccines.

Protein inclusion into ice can dissociate subunits

An antifreeze protein's inclusion into ice can be used to purify it from other proteins and solutes. Domains that are covalently attached to the antifreeze protein are also drawn into the ice such that the ice-binding portion of the fusion protein can be used as an affinity tag. Here we have explored the use of ice-affinity tags on multi-subunit proteins. When an ice-binding protein was attached as a tag to multisubunit complexes a substantial portion of each multimer dissociated during overgrowth by the ice. The protein subunit attached to the affinity tag was enriched in the ice and the other subunit was appreciably excluded. We suggest that step growth of the advancing ice front generates shearing forces on the bound complex that can disrupt non-covalent protein-protein interactions. This will effectively limit the use of ice-affinity tags to single subunit proteins.

Phylogenetic and morphological re-evaluation of Camptophora.

The genetic variety and habitats of Camptophora species, generally known as black yeast, have not been clarified. In this study, we re-evaluated Camptophora based on morphological observations and phylogenetic analyses. Because prior investigations on Camptophora only included a few strains/specimens, 24 Camptophora-related strains were newly obtained from 13 leaf samples of various plant species to redefine the genetic and species concepts of Camptophora. Their molecular phylogenetic relationships were examined using small subunit nuclear ribosomal DNA (nSSU, 18S rDNA), the internal transcribed spacer (ITS) rDNA operon, the large subunit nuclear ribosomal DNA (LSU, 28S rDNA), β-tubulin, the second largest subunit of RNA polymerase II (rpb2), and mitochondrial small subunit DNA (mtSSU). Single- and multi-locus analyses using nSSU-ITS-LSU-rpb2-mtSSU revealed a robust phylogenetic relationship among Camptophora species within Chaetothyriaceae. Camptophora species could be distinguished from other chaetothyriaceous genera by their snake-shaped conidia with microcyclic conidiation and loosely interwoven mycelial masses. Based on the results of phylogenetic analyses, two undescribed lineages were recognized, and Ca. schimae was excluded from the genus. ITS sequence comparison with environmental DNA sequences revealed that the distribution of the genus is restricted to the Asia-Pacific region. Camptophora has been isolated or detected from abrupt sources, and this was attributed to its microcycle. The mechanisms driving genetic diversity within species are discussed with respect to their phyllosphere habitats.

First report of Alternaria crassa causing yellow spots on Daturastramonium in China.

DaturastramoniumL.(jimson weed)is an invasiveweedin agricultural fieldsand a medicinalplant. In April 2022, a leaf disease on D. stramonium was observed in Zhanjiang (21.17 N, 110.18 E), Guangdong province, China. Early symptoms were small yellow spots on leaves. Later, the spots gradually expanded and turned becoming necrotic with a clear yellow halo and a white center. The disease incidence in the field was 85% (n= 50, about 1 ha). Twenty diseased leaves were collected from the field. The margin of the diseased tissues was cut into 2 mm × 2 mm pieces, surface disinfected with 75% ethanol and 2% sodium hypochlorite for 30 and 60 s, respectively, and rinsed twice with sterile water before isolation. The tissues were plated onto potato dextrose agar (PDA) medium and incubated at 28 ℃. After 2-day incubation, grayish fungal colonies appeared on the PDA, then pure cultures were produced by transferring hyphal tips to new PDA plates. Single-spore isolation method was used to recover pure cultures for three isolates (DSAC-1, DSAC-2, and DSAC-3). The isolates were morphologically identical.They colonies were gray to brownish black. Conidiophores were branched, brown. Conidia were brown, long ellipsoid, had 4-12 transverse and 0-3 longitudinal septa; measured within 67.5-127.8 (average =105.6) × 12.5-27.8 (average=20.4) µm (n=30). Apical beak was longer than conidia body. measured within 40.5-423.5 (average =365.2) × 2.5-5.8 (average=3.2) µm (n=30). Based on morphological characteristics, the three isolates were identified asAlternariacrassa(Sacc.) Rands (Simmons 2007). Molecular identification was performed using the colony polymerase chain reaction method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012) to amplify internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase second largest subunit (RPB2) and translation elongation factor (TEF1) with primers of ITS1/ITS4, GDF1/GDR1, RPB2-5F2/fRPB2-7cR, and EF-1α-F/EF-1α-R, respectively (Walther et al. 2013; Woudenberg et al. 2015; Nishikawa and Nakashima. 2020). Amplicons of the isolates were sequenced and submitted to GenBank (ITS, ON430524-ON430526; GAPDH, ON500656-ON500658; RPB2, ON500659-ON500661; TEF1, ON500662-ON500664). The sequences were 100% identical with those of Alternaria crassa strain CBS 116647 upon BLAST analysis. The sequences were also concatenated for phylogenetic analysis by maximum likelihood. The isolates clustered withA. crassa (CBS 116647, CBS 116648, CBS CBS-110.38, and CBS_103.18 ).Thus, the fungus associated with leafyellow spoton D. stramonium was identified asA. crassa.Pathogenicity tests were conducted in a greenhouse at 24℃-30℃ with 80% relative humidityusing 3 isolates. Individual plants were grown in pots (n = 5, 1 month old). The unwounded leaflets were inoculated using three isolates (DSAC-1, DSAC-2, and DSAC-3). The fungal mycelium on 5 mm-diameter PDA plugs were placed faced down to the leaves. Sterile PDA was used for mock inoculated comtrols.. The test was performed three times. Disease symptoms were observed on the leaves after 7 days, whereas the controls remained healthy. The pathogen was re-isolated from infected leaves and was morphologically identical to the original isolates, fulfilling Koch's postulates.A. crassa was reported causing leaf spot on D. stramonium in Algeria (Nabahat et al. 2020). To our knowledge, this report is the first report of A. crassa causing leaf yellow spot on D. stramonium in China. This pathogen possessespotential biocontrol properties on the invasiveweed, while this study also provides an important reference for the control of the diseaseof the medicinalplant.

The structure of Mn(II)–bound Rubisco from Spinacia oleracea

The rate of photosynthesis and, thus, CO2 fixation, is limited by the rate of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Not only does Rubisco have a relatively low catalytic rate, but it also is promiscuous regarding the metal identity in the active site of the large subunit. In Nature, Rubisco binds either Mg(II) or Mn(II), depending on the chloroplastic ratio of these metal ions; most studies performed with Rubisco have focused on Mg-bound Rubisco. Herein, we report the first crystal structure of a Mn-bound Rubisco, and we compare its structural properties to those of its Mg-bound analogues.

New species of Myxobolus in potamodromous catfish from the eastern Amazon, Brazil

The mapará (Hypophthalmus marginatus) is a commercially important fish in the Brazilian Amazon and has been described as a host for numerous myxosporid species. The integrated taxonomy of a new species, Myxobolus mickeyii n. sp., discovered in the urinary bladder of H. marginatus, is undertaken in this study. In 105 specimens of H. marginatus, plasmodia and myxospores were observed in the urinary bladder fluid, the myxospores measuring 20.5 (19.6–21.3) μm in length and 14.0 (13.2–14.9) μm in width. The posterior valves of the spore body were thick, with valvulogenic nuclei, endoplasmic reticulum, and the presence of secretory vesicles. Two elliptical, rounded appendages attached to the valve, containing tubular filaments. The two polar capsules, symmetry, measuring 6.1 (5.9–6.3) μm in length and 4.4 (3.6–6.2) μm in width, with polar tubules of 3 to 5 turns. Phylogenetic analyses of the small subunit ribosomal RNA gene (SSU rDNA) sequencing revealed that M. mickeyii n. sp. is part of a Myxobolidae family clade with freshwater fish of the Siluriformes order, with a genetic distance of 19% to the nearest species. This work contributes to the wide diversity of myxozoans in this host, as other taxa have previously been reported infecting different tissues.

A multistage protein subunit vaccine as BCG-booster confers protection against Mycobacterium tuberculosis infection in murine models

The eradication of tuberculosis remains a global challenge. Despite being the only licensed vaccine, Bacillus Calmette-Guérin (BCG) confers limited protective efficacy in adults and individuals with latent tuberculosis infections (LTBI). There is an urgent need to develop novel vaccines that can enhance the protective effect of BCG. Protein subunit vaccines have garnered significant research interest due to their safety and plasticity. Based on previous studies, we selected three antigens associated with LTBI (Rv2028c, Rv2029c, Rv3126c) and fused them with an immunodominant antigen Ag85A, resulting in the construction of a multistage protein subunit vaccine named A986. We evaluated the protective effect of recombinant protein A986 adjuvanted with MPL/QS21 as a booster vaccine for BCG against Mycobacterium tuberculosis (Mtb) infection in mice. The A986 + MPL/QS21 induced the secretion of antigen-specific Th1 (IL-2+, IFN-γ+ and TNF-α+) and Th17 (IL-17A+) cytokines in CD4+ and CD8+ T cells within the lung and spleen of mice, while also increased the frequency of central memory and effector memory T cells. Additionally, it also induced the enhanced production of IgG antibodies. Compared to BCG alone, A986 + MPL/QS21 boosting significantly augmented the proliferation of antigen-specific multifunctional T cells and effectively reduced bacterial load in infected mice. Taken together, A986 + MPL/QS21 formulation induced robust antigen-specific immune responses and provided enhanced protection against Mtb infection as a booster of BCG vaccine.

Concatemer Assisted Stoichiometry Analysis (CASA): targeted mass spectrometry for protein quantification.

Large multi-protein machines are central to multiple biological processes. However, stoichiometric determination of protein complex subunits in their native states presents a significant challenge. This study addresses the limitations of current tools in accuracy and precision by introducing concatemer-assisted stoichiometry analysis (CASA). CASA leverages stable isotope-labeled concatemers and liquid chromatography parallel reaction monitoring mass spectrometry (LC-PRM-MS) to achieve robust quantification of proteins with sub-femtomole sensitivity. As a proof-of-concept, CASA was applied to study budding yeast kinetochores. Stoichiometries were determined for ex vivo reconstituted kinetochore components, including the canonical H3 nucleosomes, centromeric (Cse4CENP-A) nucleosomes, centromere proximal factors (Cbf1 and CBF3 complex), inner kinetochore proteins (Mif2CENP-C, Ctf19CCAN complex), and outer kinetochore proteins (KMN network). Absolute quantification by CASA revealed Cse4CENP-A as a cell-cycle controlled limiting factor for kinetochore assembly. These findings demonstrate that CASA is applicable for stoichiometry analysis of multi-protein assemblies.

The Mitogenome of the Subarctic Octocoral Alcyonium digitatum Reveals a Putative tRNAPro Gene Nested within MutS.

We sequenced and analyzed the complete mitogenome of a Norwegian isolate of the octocoral Alcyonium digitatum using the Ion Torrent sequencing technology. The 18,790 bp circular mitochondrial genome was found to harbor the same set of 17 genes, which encode 14 protein subunits, two structural ribosomal RNAs and one tRNA, as reported in other octocorals. In addition, we detected a new tRNAPro-like gene sequence nested within the MutS protein coding region. This putative tRNA gene feature appears to be conserved among the octocorals but has not been reported previously. The A. digitatum mitogenome was also shown to harbor an optional gene (ORFA) that encodes a putative protein of 191 amino acids with unknown function. A mitogenome-based phylogenetic analysis, presented as a maximum likelihood tree, showed that A. digitatum clustered with high statistical confidence with two other Alcyonium species endemic to the Mediterranean Sea and the Southeast Pacific Ocean.

First DNA Barcoding Survey in Bulgaria Unveiled Huge Diversity of Yeasts in Insects.

In this study, we conducted a comprehensive survey aimed at assessing the diversity of yeast species inhabiting the guts of various insect species collected mainly from two Bulgarian National Parks, namely, Rila, and Pirin. The insect specimens encompass a broad taxonomic spectrum, including representatives from Coleoptera, Orthoptera, Lepidoptera, Hymenoptera, Dermaptera, Isopoda, and Collembola. Yeast strains were identified with DNA barcoding using the ribosomal markers, specifically, the D1/D2 domains of the ribosomal large subunit (LSU) and the internal transcribed spacers regions ITS 1 + 2 (ITS). The analysis unveiled the presence of 89 ascomycetous and 18 basidiomycetous yeast isolates associated with the insect specimens. Furthermore, our study identified 18 hitherto unknown yeast species.

Exploration of Alicyclobacillus spp. Genome in Search of Antibiotic Resistance.

The study investigates the antibiotic resistance (AR) profiles and genetic determinants in three strains of guaiacol-producing Alicyclobacillus spp. isolated from orchard soil and pears. Their phenotypic characteristics, such as spore formation; resistance to different factors, including drugs or disinfectants; or production of off-flavor compounds, can affect the taste and aroma of spoiled products. Food and beverages are potential vectors for the transfer of antibiotic resistance genes, which is a growing health concern; thus, microorganisms in food and beverages should not be a potential source of drug resistance to consumers. Whole-genome sequencing (WGS) was utilized to identify antibiotic resistance genes, metabolic pathways, and elements associated with guaiacol and halophenol production. Minimum inhibitory concentration (MIC) testing revealed that all strains were susceptible to eight out of nine tested antibiotics (ampicillin, gentamycin, kanamycin, streptomycin, clindamycin, tetracycline, chloramphenicol, and vancomycin) but exhibited high resistance to erythromycin. Analysis indicated that the erythromycin resistance gene, ribosomal RNA small subunit methyltransferase A (RsmA), was intrinsic and likely acquired through horizontal gene transfer (HGT). The comprehensive genomic analysis provides insights into the molecular mechanisms of antibiotic resistance in Alicyclobacillus spp., highlighting the potential risk of these bacteria as vectors for antibiotic resistance genes in the food chain. This study expands the understanding of the genetic makeup of these spoilage bacteria and their role in antimicrobial resistance dissemination.

An insight into the cryptic diversity of Fragilariaceae (Bacillariophyta), with the description of a new Antarctic species, Gedaniella antarctica sp. nov.

ABSTRACT Fragilariaceae is a paraphyletic family of araphid diatoms commonly used as bio-indicators, in environmental assessments and in paleoenvironmental reconstructions, and with various potential industrial applications. Recent molecular taxonomic research has highlighted significant limitations in traditional morphology-based investigations of these diatom species. Most descriptions of species and genera of the Fragilariaceae present broad morphological character definitions and many diagnostic characters are indistinguishable by light microscopy. In this sense, taxon misidentification is common and could have serious implications for environmental surveys and laboratory experiments. To better understand the diversity of the Fragilariaceae, we (1) performed phylogenetic analyses of the small subunit ribosomal RNA (18S rRNA), rbcL and psbC gene sequences, (2) inferred a cladogram from key morphological features used in the traditional identification of Fragilariaceae, (3) tested if the topologies of the tree recovered from the molecular phylogeny, of the tree based on morphology and of the trees constraining to monophyly the genera Sarcophagodes, Pseudostaurosira and Nanofrustulum (together and separately) were significantly different and (4) mapped morphological character states on the ML tree and inferred their evolution based on maximum parsimony. Our results supported the monophyly of a group of Fragilariaceae within small araphid diatoms including the genera Cratericulifera, Plagiostriata, Castoridens, Opephora, Staurosira, Staurosirella, Punctastriata, Psammotaenia, Hendeyella, Stauroforma, Pseudostaurosira sensu Li, Nanofrustulum sensu Li, Serratifera sensu Li and Gedaniella sensu Li. Molecular phylogeny and topology tests suggested that the latest circumscriptions of the genera Sarcophagodes, Pseudostaurosira and Nanofrustulum sensu Morales were not monophyletic. Analyses of the Antarctic strain IMA070A collected during the XXXIV Italian Antarctic Expedition using fine structural features of the frustule and molecular data revealed that this diatom belongs to a distinct lineage within Gedaniella, which we describe here as Gedaniella antarctica sp. nov.

Miamiensis avidus, a Novel Scuticociliate Pathogen Isolated and Identified from Cultured Large Yellow Croaker (Larimichthys crocea).

Scuticociliates are recognized as the causative agents of mass mortalities in certain cultured marine fishes, resulting in enormous economic losses. This study aimed to investigate a fatal infection caused by scuticociliates in farmed large yellow croaker (Larimichthys crocea) in Fujian province, China. Microscopic examinations of focal organs, including the brain, eyes, gills, and skin, revealed the presence of parasites. Active masses of scuticociliates were observed in these organs, and the ciliates were subsequently isolated and maintained in vitro. An immersion challenge experiment revealed that L. crocea experienced cumulative mortalities reaching 73% within 7 d upon exposure to 1.0 × 104 ciliates mL-1. Based on the microscopic and PCR testing of infected fishes, the brain was comprehensively inferred as the main infection organ for the isolated strain. Microscopic and submicroscopic observations of the isolated scuticociliate, coupled with cortical ciliature patterns revealed through α-tubulin indirect immunofluorescence techniques, identified these scuticociliates as Miamiensis avidus. The sequencing of two genetic markers (small subunit ribosomal RNA, SSU rRNA and cytochrome c oxidase subunit I, COI) further confirmed that the isolated strains exhibited the highest sequence similarity to most M. avidus sequences in GenBank. However, significant differences in SSU sequences compared to the M. avidus strain Ma/2, and the lack of published COI and ITS (internal transcribed spacer) sequences for Ma/2, indicate the need for further molecular data to resolve whether there are potential cryptic species within the M. avidus complex.