Domain Of Subunit Research Articles

Vishniacozyma siamensis sp. nov., a new anamorphic tremellomycetous yeast species isolated from a mangrove forest in Thailand.

Six strains (DMKU-SG26, DMKU-SG42, DMKU-SYM22, DMKU-RG41, DMKU-RX317 and DMKU-RGM25) representing a novel basidiomycetous yeast species were isolated from leaf surfaces of mangrove plants collected in Thailand. Pairwise sequence analysis indicated that the six strains either had identical nucleotide substitution in the D1/D2 domains of the large subunit (LSU) rRNA gene sequences or differed by one to three nucleotide(s). They also had identical or differed by one to five nucleotide substitution(s) in the internal transcribed spacer (ITS) regions. blastn searches of the GenBank database revealed that the six strains were closely related to the holotype of type strains of Vishniacozyma peneaus, V. terrae, V. phoenicis, V. taiwanica and V. europaea, but with 6-15 (1.14-2.48%) and 16-26 (5.4-8.8%) nucleotide substitutions in the D1/D2 domains of the LSU rRNA gene and the ITS regions, respectively. Phylogenetic analysis based on the concatenated sequences of the ITS regions and D1/D2 domains of the LSU rRNA gene showed that these strains are placed in the Vishniacozyma clade but were at a distinctly different position from the other recognized species of the genus. Based on the phylogenetic analysis and phenotypic characteristics, these six strains are a novel species of the genus Vishniacozyma, for which the name Vishniacozyma siamensis sp. nov. is proposed to accommodate them. The holotype is TBRC 18499T and the ex-type culture is PYCC 10042 (=DMKU-SG26). The MycoBank number of the novel species is MB 855838.

Conformational response of αIIbβ3 and αVβ3 integrins to force.

As major adhesion receptors, integrins transmit biochemical and mechanical signals across the plasma membrane. These functions are regulated by transitions between bent and extended conformations and modulated by force. To understand how force on integrins mediates cellular mechanosensing, we compared two highly homologous integrins, αIIbβ3 and αVβ3. These integrins, expressed in circulating platelets vs. solid tissues, respectively, share the β3 subunit, bind similar ligands and have similar bent and extended conformations. Here, we report that in cells expressing equivalent levels of each integrin, αIIbβ3 mediates spreading on softer substrates than αVβ3. These effects correlate with differences in structural dynamics of the two integrins under force. All-atom simulations show that αIIbβ3 is more flexible than αVβ3 due to correlated residue motions within the α subunit domains. Single molecule measurements confirm that αIIbβ3 extends faster than αVβ3. These results reveal a fundamental relationship betweenprotein function and structural dynamics in cell mechanosensing.

Meira qingyuanensis sp. nov., an anamorphic yeast species isolated from plant leaves.

Two yeast strains, NYNU 224191 and NYNU 22411, were isolated from plant leaves of Cyclobalanopsis jenseniana and Schisandra sp. harvested from Qingyuan Mountain, Quanzhou City, Fujian Province, southeast China. Molecular phylogenetic analyses demonstrated that these two strains represent a novel species in the genus Meira. They are more closely related to M. marina, M. nashicola, M. plantarum and M. pileae than other Meira species based on phylogenetic analysis of the internal transcribed spacer (ITS) region and the D1/D2 domain of the large subunit (LSU) rRNA gene. The novel species differed from its closest relatives, M. marina, M. nashicola, M. plantarum and M. pileae, by 1.8-2% sequence divergence (11-12 substitutions and 1-2 gaps) in the D1/D2 domain and over 6.7% sequence divergence (40-60 substitutions and 53-77 gaps) in the ITS region, respectively. We propose the name Meira qingyuanensis sp. nov. (Holotype CICC 33567; Mycobank MB 855328) for the new species, which differs phenotypically from M. marina, M. plantarum and M. pileae in its ability to assimilate methyl-α-d-glucoside and the inability to assimilate salicin, and from M. nashicola in its ability to assimilate nitrate and nitrite and grow at 30 °C.

Tripartite interactions of PKA catalytic subunit and C-terminal domains of cardiac Ca2+ channel may modulate its β-adrenergic regulation

BackgroundThe β-adrenergic augmentation of cardiac contraction, by increasing the conductivity of L-type voltage-gated CaV1.2 channels, is of great physiological and pathophysiological importance. Stimulation of β-adrenergic receptors (βAR) activates protein kinase A (PKA) through separation of regulatory (PKAR) from catalytic (PKAC) subunits. Free PKAC phosphorylates the inhibitory protein Rad, leading to increased Ca2+ influx. In cardiomyocytes, the core subunit of CaV1.2, CaV1.2α1, exists in two forms: full-length or truncated (lacking the distal C-terminus (dCT)). Signaling efficiency is believed to emanate from protein interactions within multimolecular complexes, such as anchoring PKA (via PKAR) to CaV1.2α1 by A-kinase anchoring proteins (AKAPs). However, AKAPs are inessential for βAR regulation of CaV1.2 in heterologous models, and their role in cardiomyocytes also remains unclear.ResultsWe show that PKAC interacts with CaV1.2α1 in heart and a heterologous model, independently of Rad, PKAR, or AKAPs. Studies with peptide array assays and purified recombinant proteins demonstrate direct binding of PKAC to two domains in CaV1.2α1-CT: the proximal and distal C-terminal regulatory domains (PCRD and DCRD), which also interact with each other. Data indicate both partial competition and possible simultaneous interaction of PCRD and DCRD with PKAC. The βAR regulation of CaV1.2α1 lacking dCT (which harbors DCRD) was preserved, but subtly altered, in a heterologous model, the Xenopus oocyte.ConclusionsWe discover direct interactions between PKAC and two domains in CaV1.2α1. We propose that these tripartite interactions, if present in vivo, may participate in organizing the multimolecular signaling complex and fine-tuning the βAR effect in cardiomyocytes.

Design and functional validation of a chimeric E3 ubiquitin ligase targeting the spike protein S1 subunit of SARS-CoV-2

The spike (S) protein plays a crucial role in the entry of SARS-CoV-2 into host cells. The S protein contains two subunits, S1 and S2. The receptor-binding domain (RBD) of the S1 subunit binds to the receptor angiotensin-converting enzyme 2 (ACE2) to enter the host cells. Therefore, degrading S1 is one of the feasible strategies to inhibit SARS-CoV-2 infection. The purpose of this study is to develop a degradation tool targeting S1. First, we constructed a HEK 293 cell line stably expressing S1 by using a three-plasmid lentivirus system. The overexpression of the mitochondrial E3 ubiquitin protein ligase 1 (MUL1) in this cell line promoted the ubiquitination of S1 and accelerated its proteasomal degradation. Further research showed the polyubiquitination of S1 catalyzed by MUL1 mainly occurred via the addition of K48-linked chains. Moreover, the specific peptide LCB1, which targets and recognizes S1, was combined with MUL1 to create the chimeric E3 ubiquitin ligase LCB1-MUL1. In comparison to MUL1, this chimeric enzyme demonstrated improved catalytic efficiency, resulting in a reduction of S1's half-life from 12 h to 9 h. In summary, this study elucidated the mechanism by which MUL1 promotes the ubiquitination modification of S1 and facilitates its degradation through the proteasome, and preliminarily validated the effectiveness of targeted degradation of S1 by chimeric enzyme LCB1-MUL1.

Agonist activation to open the Gα subunit of the GPCR–G protein precoupled complex defines functional agonist activation of TAS2R5

G protein-coupled receptors (GPCRs) regulate multiple cellular responses and represent highly successful therapeutic targets. The mechanisms by which agonists activate the G protein are unclear for many GPCR families, including the bitter taste receptors (TAS2Rs). We ascertained TAS2R5 properties by live cell-based functional assays, direct binding affinity measurements using optical resonators, and atomistic molecular dynamics simulations. We focus on three agonists that exhibit a wide range of signal transduction in cells despite comparable ligand-receptor binding energies derived from direct experiment and computation. Metadynamics simulations revealed that the critical barrier to activation is ligand-induced opening of the G protein between the α-helical (AH) and Ras-like domains of Gα subunit from a precoupled TAS2R5-G protein state to the fully activated state. A moderate agonist opens the AH-Ras cleft from 22 Å to 31 Å with an energy gain of -4.8 kcal mol-1, making GDP water-exposed for signaling. A high-potency agonist had an energy gain of -11.1 kcal mol-1. The low-potency agonist is also exothermic for Gα opening, but with an energy gain of only -1.4 kcal mol-1. This demonstrates that TAS2R5 agonist-bound functional potencies are derived from energy gains in the transition from a precoupled complex at the level of Gα opening. Our experimental and computational study provides insights into the activation mechanism of signal transduction that provide a basis for rational design of new drugs.

An in-silico study reveals that a C-terminal fragment of the adhesion protein Fibulin7 (Fbln7-C) regulates the activation of integrin α5β1 through dynamics of VWA and the hybrid domain in the β1 subunit

A C-terminal fragment of the adhesion protein Fibulin7 (Fbln7-C) binds to monocytes and neutrophils via integrin α5β1, regulating their adhesion and immunological functions through Erk and STAT signaling pathways. It also inhibits cell binding, spreading, and migration on fibronectin. However, the precise structural components of Fbln7-C that interact with various domains of integrin α5β1 and contribute to its regulatory effects are not entirely understood. This study investigated the structural dynamics of Fibulin7 fragments and the mechanisms by which Fbln7-C regulates α5β1 integrin activation using protein modeling, protein-protein docking, molecular dynamics simulation (MDS), and binding free energy calculations. An energy-minimized model of α5β1 integrin, Fibulin7 full length (Fbln7-FL), and Fbln7-C was developed and validated using 100 ns MDS. Additionally, protein-protein docking was used to confirm Fbln7-C’s better integrin binding ability over Fbln7-FL. A 500 ns MDS on the docked Fbln7-C integrin complex revealed the regulatory effects of Fbln7-C on arginine-glycine-aspartic acid (RGD) bound integrin α5β1. The simulation studies showed that Fbln7-C’s attachment to activated α5β1 integrin increased the distance between the RGD and its interacting residues on both integrin subunits, shifting the RGD ligand from its original binding position and inactivating the integrin. Further analysis using free energy landscape (FEL), principal component analysis (PCA), and binding energy calculation validated the alteration in α5β1 integrin’s structural dynamics following Fbln7-C binding. This could relate to obstruction in the outward swing of the integrin’s hybrid domain and result in the low-affinity, inactive headpiece conformation of the α5β1 integrin.

Vanderwaltozyma japonica sp. nov.: a novel yeast isolated from subtropical regions in Japan.

Seven yeast strains, NBRC 11072, NBRC 11076, NBRC 11078T, NBRC 11080, NBRC 116645, NBRC 116646 and NBRC 116647, were isolated from the subtropical regions of Japan. The seven strains share identical nucleotide sequences in the D1/D2 domains of the large subunit (LSU) rRNA gene and internal transcribed spacer (ITS) regions. Six strains (NBRC 11072, NBRC 11076, NBRC 11078T, NBRC 11080, NBRC 116645 and NBRC 116646) share identical mitochondrial cytochrome c oxidase subunit II (COX II) sequences as well, while NBRC 116647 differs from them by one substitution in this region. The yeast strains were closely related to Vanderwaltozyma verrucispora NBRC 1884T in the ITS regions. However, in the D1/D2 domains and COX II, they were closely related to Torulaspora globosa CBS 764T and Naumovozyma castellii NRRL Y-12630T, respectively. They differed from V. verrucispora by 49 nucleotide substitutions with 26 gaps in the ITS region, from T. globosa by 19 nucleotide substitutions with 3 gaps in the D1/D2 domains and from N. castellii by 49 nucleotide substitutions with 6 gaps in the COX II. The phylogenetic analysis of the seven strains based on the sequences of the D1/D2 domains of LSU and ITS regions among related species showed that these strains represent a single novel species within the genus Vanderwaltozyma. The seven strains could be differentiated from other known species of the genus Vanderwaltozyma because of their inability to assimilate glycerol as a carbon source. Therefore, we suggest that these seven strains represent a novel species, designated Vanderwaltozyma japonica sp. nov. The holotype is NBRC 11078T (isotype: TBRC 19102T). The MycoBank number is MB 855476.

Possible rapid reduction of anti-RBD antibody titre after SARS-CoV-2 mRNA vaccination in pregnant women: Multicentre prospective study.

Pregnant women are at increased risk for severe illness associated with coronavirus disease 2019 (COVID-19) compared to nonpregnant women. The aim of this multicenter prospective study was to assess the current COVID-19 vaccination status of pregnant women in the southern Osaka district and to compare their antibody titers with those of nonpregnant women. Serum antibody titers of anti-NCP antibodies (antibodies against the SARS-CoV-2 nucleocapsid) and anti-RBD antibodies (the receptor binding domain of the S1 subunit of the spike protein) were evaluated in 753 pregnant women at 34-35 weeks of gestation from October 2021 to March 2022. Anti-RBD antibody titre was also investigated in 1003 health care workers at Kindai University hospital 3 and 6 months after a second dose of the vaccine from March 2021 to April 2021. 519 (68.9%) pregnant women were vaccinated during pregnancy, of whom 497 (95.8%) received two doses. The COVID-19 infection rate calculated from the number of pregnant women with a positive anti-NCP antibody titre or with confirmed diagnosis was 5.1% (12/234) in the unvaccinated and 3.5% (18/519) in the vaccinated. The estimated half-life calculated from anti-RBD antibody titers and the number of days between vaccination and antibody testing was 39.9 days. The antibody titre and half-life in pregnant women were significantly lower and shorter than in nonpregnant women aged 20-39 years (109.4 days). Our study showed that pregnant women may have lower vaccine-acquired COVID-19 immunity than nonpregnant women.

The BAF complex enhances transcription through interaction with H3K56ac in the histone globular domain

Histone post-translational modifications play pivotal roles in eukaryotic gene expression. To date, most studies have focused on modifications in unstructured histone N-terminal tail domains and their binding proteins. However, transcriptional regulation by chromatin-effector proteins that directly recognize modifications in histone globular domains has yet to be clearly demonstrated, despite the richness of their multiple modifications. Here, we show that the ATP-dependent chromatin-remodeling BAF complex stimulates p53-dependent transcription through direct interaction with H3K56ac located on the lateral surface of the histone globular domain. Mechanistically, the BAF complex recognizes nucleosomal H3K56ac via the DPF domain in the DPF2 subunit and exhibits enhanced nucleosome-remodeling activity in the presence of H3K56ac. We further demonstrate that a defect in H3K56ac–BAF complex interaction leads to impaired p53-dependent gene expression and DNA damage responses. Our study provides direct evidence that histone globular domain modifications participate in the regulation of gene expression.

Absence of the Third Linker Domain of ApcE Subunit in Phycobilisome from Synechocystis 6803 Reduces Rods-To-Core Excitation Energy Transfer.

Phycobilisome (PBS) is a pigment-protein complex utilized by red algae and cyanobacteria in photosynthesis for light harvesting. A cyanobacterium Synechocystis sp. PCC 6803 contains PBS with a tricylindrical core built of allophycocyanin (APC) disks where six phycocyanin (PC) rods are attached. The top core cylinder is seemingly involved in attaching four PC rods and binding orange carotenoid protein (OCP) to quench excess of excitation energy. In this study, we have deleted the third linker domain (LD3) of ApcE subunit of PBS which assembles four APC discs into the top core cylinder. The mutation resulted in PBS with bicylindrical core, structurally comparable to the naturally existing PBS from Synechococcus 7942. Lack of LD3 and the top APC cylinder reduces the excitation energy transfer between PC and APC in the mutant. Moreover, these PBSs are more prone to light induced-photodamage and do not bind to the photoactivated orange carotenoid protein (OCP), a known PBS excitation quencher. These findings highlight the complex and elegant interplay between the PBS architecture and the functional efficiency, suggesting that in PBSs with naturally tri-cylindrical cores, the top cylinder has essential roles in recruiting the rods and proper binding of OCP.

Rapid and efficient immune response induced by a designed modular cholera toxin B subunit (CTB)-based self-assembling nanoparticle

Modular self-assembling nanoparticle vaccines, represent a cutting-edge approach in immunology with the potential to revolutionize vaccine design and efficacy. Although many innovative efficient modular self-assembling nanoparticles have been designed for vaccination, the immune activation characteristics underlying such strong protection remain poorly understood, limiting the further expansion of such nanocarrier. Here, we prepared a novel modular nanovaccine, which self-assembled via a pentamer cholera toxin B subunit (CTB) domain and an unnatural trimer domain, presenting S. Paratyphi A O-polysaccharide antigen, and investigated its rapid immune activation mechanism. The nanovaccine efficiently targets draining lymph nodes and antigen-presenting cells, facilitating co-localization with Golgi and endoplasmic reticulum. In addition, dendritic cells, macrophages, B cells, and neutrophils potentially participate in antigen presentation, unveiling a dynamic change of the vaccines in lymph nodes. Single-cell RNA sequencing at early stage and iN vivo/iN vitro experiments reveal its potent humoral immune response capabilities and protection effects. This nanoparticle outperforms traditional CTB carriers in eliciting robust prophylactic effects in various infection models. This work not only provides a promising and efficient candidate vaccine, but also promotes the design and application of the new type of self-assembled nanoparticle, offering a safe and promising vaccination strategy for infection diseases.

Clinical phenotype and functional influence of GRIN2A variants in epilepsy-aphasia syndrome.

N-methyl-D-aspartate receptors are glutamate-gated ion channels that play a crucial role in brain function. Numerous inherited or de novo variants in the GRIN2A gene, encoding the GluN2A subunit of the receptor, have been identified in patients with epilepsy. In addition, it is worth noting that GRIN2A variants exhibit a strong correlation with epilepsy-aphasia syndromes, a group of age-dependent epileptic, cognitive, and language disorders with a characteristic electroencephalographic pattern. Whole exome sequencing was conducted in enrolled patients with epilepsy-aphasia syndromes, and GRIN2A variants were screened. The conservation of substituted residues, conformational changes of mutant subunits, and in silico predictions of pathogenicity were thoroughly assessed in our study. Functional alterations of the variants were examined using whole-cell voltage-clamp current recordings while the relative surface expression levels of subunit proteins were assessed via immunofluorescence assays. A summary of previously published GRIN2A missense variants was conducted to investigate the genotypic-phenotypic-functional correlations. Two missense GRIN2A variants (c. 2482A >G/p. M828V, c.2627 T >C/p. I876T) were identified, which are located in the transmembrane helix M4 and C-terminus domain of the GluN2A subunit, respectively. Both variants exhibited reduced current density of NMDARs and surface/total expression levels of GluN2A subunits, while M828V showed a decreased extent of desensitization as well. A further summary of the previously reported GRIN2A variants demonstrated that more variable phenotypes were observed for variants situated in the C-terminus domain or those with loss-of-function effects. Our study expands the phenotypic and functional range of GRIN2A-related disorders. In order to optimally establish the domain-function-phenotype correlations in GRIN2A variants, it is imperative to gather a more extensive set of clinical and functional data. This study has identified two genetic variants of the GRIN2A gene in patients with epilepsy-aphasia syndrome. We assess the variants' harmfulness through a variety of functional experiments, including evaluating the expression level of the mutated protein and the resulting changes in electrophysiological activities. Also, we reviewed previously published papers about GRIN2A variants in epilepsy to learn more about the correlations between their locations, functional changes, and clinical manifestations.

Sequence and structural determinants of RNAPII CTD phase-separation and phosphorylation by CDK7

The intrinsically disordered carboxy-terminal domain (CTD) of the largest subunit of RNA Polymerase II (RNAPII) consists of multiple tandem repeats of the consensus heptapeptide Y1-S2-P3-T4-S5-P6-S7. The CTD promotes liquid-liquid phase-separation (LLPS) of RNAPII in vivo. However, understanding the role of the conserved heptad residues in LLPS is hampered by the lack of direct biochemical characterization of the CTD. Here, we generated a systematic array of CTD variants to unravel the sequence-encoded molecular grammar underlying the LLPS of the human CTD. Using in vitro experiments and molecular dynamics simulations, we report that the aromaticity of tyrosine and cis-trans isomerization of prolines govern CTD phase-separation. The cis conformation of prolines and β-turns in the SPXX motif contribute to a more compact CTD ensemble, enhancing interactions among CTD residues. We further demonstrate that prolines and tyrosine in the CTD consensus sequence are required for phosphorylation by Cyclin-dependent kinase 7 (CDK7). Under phase-separation conditions, CDK7 associates with the surface of the CTD droplets, drastically accelerating phosphorylation and promoting the release of hyperphosphorylated CTD from the droplets. Our results highlight the importance of conformationally restricted local structures within spacer regions, separating uniformly spaced tyrosine stickers of the CTD heptads, which are required for CTD phase-separation.

The accomplices: Heparan sulfates and N-glycans foster SARS-CoV-2 spike:ACE2 receptor binding and virus priming

Although it is well established that the SARS-CoV-2 spike glycoprotein binds to the host cell ACE2 receptor to initiate infection, far less is known about the tissue tropism and host cell susceptibility to the virus. Differential expression across different cell types of heparan sulfate (HS) proteoglycans, with variably sulfated glycosaminoglycans (GAGs), and their synergistic interactions with host and viral N-glycans may contribute to tissue tropism and host cell susceptibility. Nevertheless, their contribution remains unclear since HS and N-glycans evade experimental characterization. We, therefore, carried out microsecond-long all-atom molecular dynamics simulations, followed by random acceleration molecular dynamics simulations, of the fully glycosylated spike:ACE2 complex with and without highly sulfated GAG chains bound. By considering the model GAGs as surrogates for the highly sulfated HS expressed in lung cells, we identified key cell entry mechanisms of spike SARS-CoV-2. We find that HS promotes structural and energetic stabilization of the active conformation of the spike receptor-binding domain (RBD) and reorientation of ACE2 toward the N-terminal domain in the same spike subunit as the RBD. Spike and ACE2 N-glycans exert synergistic effects, promoting better packing, strengthening the protein:protein interaction, and prolonging the residence time of the complex. ACE2 and HS binding trigger rearrangement of the S2' functional protease cleavage site through allosteric interdomain communication. These results thus show that HS has a multifaceted role in facilitating SARS-CoV-2 infection, and they provide a mechanistic basis for the development of GAG derivatives with anti-SARS-CoV-2 potential.

Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

Prenatal SARS-CoV-2 infection is associated with higher rates of pregnancy and birth complications, despite that vertical transmission rates are thought to be low. Here, multi-omics analyses of human placental tissues, cord tissues/plasma, and amniotic fluid from 23 COVID-19 mother-infant pairs revealed robust inflammatory responses in both maternal and fetal compartments. Pronounced expression of complement proteins (C1q, C3, C3b, C4, C5) and inflammatory cytokines (TNF, IL-1α, and IL-17A/E) was detected in the fetal compartment of COVID-19-affected pregnancies. While ~26% of fetal tissues were positive for SARS-CoV-2 RNA, more than 60% of fetal tissues contained SARS-CoV-2 ORF8 proteins, suggesting transplacental transfer of this viral accessory protein. ORF8-positive fetal compartments exhibited increased inflammation and complement activation compared to ORF8-negative COVID-19 pregnancies. In human placental trophoblasts in vitro, exogenous ORF8 exposure resulted in complement activation and inflammatory responses. Co-immunoprecipitation analysis demonstrated that ORF8 binds to C1q specifically by interacting with a 15-peptide region on ORF8 (C37-A51) and the globular domain of C1q subunit A. In conclusion, an ORF8-C1q-dependent complement activation pathway was identified in COVID-19-affected pregnancies, likely contributing to fetal inflammation independently of fetal virus exposure.

B-050 A comparison of two semi-quantitative tests, Abbott’s Advise DX SARS-CoV-2 IgG II and Beckman Coulter’s Access SARS-COV-2 IgG II, for measuring IgG antibody levels after Sars-CoV-2 Pfizer-BioNTech vaccination

Abstract Background Using 219 samples collected from volunteer employees of our lab, obtained in different times after Sars-CoV-2 Pfizer-BioNTech vaccination, we compared two semi-quantitative tests: Methods Abbott's AdviseDx SARS-CoV-2 IgG II and Beckman Coulter’s Access SARS-CoV-2 IgG II assay. Both assays detect IgG antibodies to the receptor binding domain of the S1 subunit of the spike protein of SARS-CoV-2 in serum and plasma of tested individuals. For the Beckman test, AMR is from 0 to 450 AU/ml with cut off for positive test 10 AU/ml; for the Abbott test, AMR is from 0 to 25000 AU/ml with cut off for positive test 100 AU/ml. Results Using a fitted regression line, it appears that the best correlation between these two tests was found in the lower value range, below 25AU/ml for Beckman test and 4000 AU/ml for Abbott test (R-squared value of 0.842 and y=87.165x). For higher antibody levels, the result distribution resembles a binary pattern, which we find very interesting. We will discuss possible explanations of this finding. Conclusions

Characterization and biocontrol potential of Wickerhamomyces subpelliculosus yeasts isolated from dates: Volatile compounds-mediated antifungal activity against mycotoxigenic Penicillium strains

Seven yeast strains were isolated from Tunisian dates. The strains were identified by sequence analysis of the D1/D2 domain of the nuclear large subunit (LSU) rRNA gene. Based on this all strains in the study were almost identical with that of the type strain of Wickerhamomyces subpelliculosus (CBS 5767) indicating that they belong to this species. All strains were characterized physiologically and biochemically. All strains grew in the presence of 50 % sucrose, 10 % sodium chloride and at 42 °C. The potential of these yeasts as biocontrol agent against mycotoxigenic Penicillium species inhabiting date, was evaluated. All yeast strains inhibited the growth of P. citrinum P10 and P. chrysogenum C17 previously isolated from dates, with inhibition percentages ranging between 43.6 % and 70.3 % on dual culture plate assays. Moreover, the volatile compounds (VCs) produced by these yeasts inhibited the mycelial growth rate and sporulation of both fungus strains, up to 76.5 and 100 %, respectively, on inverted culture plate assay. The VCs of W. subpelliculosus strains Y4 and Y24, which exhibit strong inhibitory activity against toxigenic Penicillium, were determined by head-space solid-phase microextraction (HS-SPME) combined with gas chromatography coupled with mass spectrometry (GC–MS) analysis. Results revealed significant levels of alcohols (27.36 % for Y4 and 23.35 % for Y24) and esters (66.19 % for Y4 and 75.82 % for Y24). Their significant bioactivity, along with the lack of reported adverse effects on consumer health or the environment, makes them a sustainable and effective alternative to synthetic fungicides for the biocontrol of mycotoxigenic Penicillium affecting stored dates.

AraC Functional Suppressors of Mutations in the C-Terminal Domain of the RpoA Subunit of the Escherichia coli RNA Polymerase.

In E. coli, transcriptional activation is often mediated by the C-terminal domain of RpoA, the α subunit of RNA polymerase. Random mutations that prevent activation of the arabinose PBAD promoter are clustered in the RpoA C-terminal domain (α-CTD). We have isolated functional suppressors of rpoA α-CTD mutations that map to araC, the main transcriptional regulator of ara genes, or to the PBAD promoter. No mutation was found in the DNA regulatory region between araC and PBAD. Most suppressors that improve PBAD transcription are localized to the N-terminal domain of AraC. One class of araC mutations generates substitutions in the core of the N-terminal domain, suggesting that they affect its conformation. Other suppressors localize to the flexible N-terminal arm of AraC. Some, but not all, suppressors confer an arabinose constitutive phenotype. Suppression by both classes of araC mutations requires the α-CTD to stimulate expression from PBAD. Surprisingly, in rpoA+ strains lacking Crp, the cAMP receptor protein, these araC mutations largely restore arabinose gene expression and can essentially bypass Crp activation. Thus, the N-terminal domain of AraC exhibits at least three distinct activities: dimerization, arabinose binding, and transcriptional activation. Finally, one mutation maps to the AraC C-terminal domain and can synergize with AraC mutations in the N-terminal domain.

PIP2 inhibits pore opening of the cyclic nucleotide-gated channel SthK

The signaling lipid phosphatidylinositol-4,5-bisphosphate (PIP2) regulates many ion channels. It inhibits eukaryotic cyclic nucleotide-gated (CNG) channels while activating their relatives, the hyperpolarization-activated and cyclic nucleotide-modulated (HCN) channels. The prokaryotic SthK channel from Spirochaeta thermophila shares features with CNG and HCN channels and is an established model for this channel family. Here, we show SthK activity is inhibited by PIP2. A cryo-EM structure of SthK in nanodiscs reveals a PIP2-fitting density coordinated by arginine and lysine residues from the S4 helix and the C-linker, located between voltage-sensing and pore domains of adjacent subunits. Mutation of two arginine residues weakens PIP2 inhibition with the double mutant displaying insensitivity to PIP2. We propose that PIP2 inhibits SthK by gluing S4 and S6 together, stabilizing a resting channel conformation. The PIP2 binding site is partially conserved in CNG channels suggesting the possibility of a similar inhibition mechanism in the eukaryotic homologs.