Biology subjects

Lee, K. H.

Publications and source records attributed to Lee, K. H..

At least 37 records · Page 2Linked to original sources

bioRxiv2022

CD244 regulates both innate and adaptive immune axes in melanoma by inhibiting autophagy-mediated M1 macrophage maturation

Accumulating data have highlighted the role of monocytes/macrophages in immune escape by generating immunologically "cold" tumors that do not respond to immunotherapy. CD244 (SLAMF4, 2B4), a member of the signaling lymphocyte activation molecule family, is expressed on myeloid cells, but its precise role has not been elucidated. Using monocyte lineage-specific CD244-deficient (LysM-cre+/-CD244fl/fl;cKO) mice challenged with B16F10 melanoma, we report for the first time that CD244 negatively regulates tumor immunity by inhibiting the differentiation and functional maturation of CD11b+Ly6ChiF4/80lo monocytes into CD11b+Ly6CloF4/80hi macrophages within the tumor microenvironment. CD244-deficient macrophages more effectively activated antigen-specific T cell responses compared to WT macrophages, thus delaying tumor growth in the B16F10 melanoma model. Moreover, combinatorial intervention of anti-PD-L1 antibodies with CD244-KO BMDM markedly improved tumor rejection compared to the anti-PD-L1 antibody alone or in combination with WT BMDM. Consistent with the murine data, transcriptome analysis of human melanoma tissue single-cell RNA-sequencing dataset (SCP398 from single-cell portal), revealed 221 differentially expressed genes of CD244- monocytes/macrophages were associated with phagocytosis, antigen presentation, and autophagy. Additionally, cell type deconvolution analysis within melanoma patients bulk RNA-seq datasets from TCGA database, revealed presence of CD244- monocytes/macrophages significantly increased patient survival in primary and metastatic tumors. Hence, we proposed that CD244 serve as a critical immune checkpoint receptor on macrophages, and CD244-deficient macrophages may represent a novel therapeutic modality to convert immunologically "cold" tumors to "hot" tumors, which can function synergistically with checkpoint blockade therapies.

immunology↗

bioRxiv2022

Identification and Characterization of ML321: a Novel and Highly Selective D2 Dopamine Receptor Antagonist with Efficacy in Animal Models that Predict Atypical Antipsychotic Activity

We have developed and characterized a novel D2R antagonist with exceptional GPCR selectivity - ML321. In functional profiling screens of 168 different GPCRs, ML321 showed little activity beyond potent inhibition of the D2R, and to a lesser extent the D3R, demonstrating excellent receptor selectivity. The D2R selectivity of ML321 may be related to the fact that, unlike other monoaminergic ligands, ML321 lacks a positively charged amine group and adopts a unique binding pose within the orthosteric binding site of the D2R. PET imaging studies in non-human primates demonstrated that ML321 penetrates the CNS and occupies the D2R in a dose-dependent manner. Behavioral paradigms in rats demonstrate that ML321 can selectively antagonize a D2R-mediated response (hypothermia) while not affecting a D3R-mediated response (yawning) using the same dose of drug, thus indicating exceptional in vivo selectivity. We also investigated the effects of ML321 in animal models that are predictive of antipsychotic efficacy in humans. We found that ML321 attenuates both amphetamine- and phencyclidine-induced locomotor activity and restored pre-pulse inhibition (PPI) of acoustic startle in a dose-dependent manner. Surprisingly, using doses that were maximally effective in both the locomotor and PPI studies, ML321 was relatively ineffective in promoting catalepsy. Kinetic studies revealed that ML321 exhibits slow-on and fast-off receptor binding rates, similar to those observed with atypical antipsychotics with reduced extrapyramidal side effects. Taken together, these observations suggest that ML321, or a derivative thereof, may exhibit "atypical" antipsychotic activity in humans with significantly fewer side effects than observed with currently FDA-approved D2R antagonists.

pharmacology and toxicology↗

bioRxiv2022

Unusual Morphological Changes of Rugositalea oryzae, A Novel Wrinkled Bacterium Isolated from The Rice Rhizosphere, Under Nutrient Stress

Bacterial cell morphology might result from natural selection to gain a competitive advantage under environmentally stressful conditions such as nutrient limitation. A bacterial strain YC6860T isolated from the rhizosphere of rice (Oryza sativa L.) showed pleomorphic behavior with smooth cell morphology and wrinkled surface rods depending upon nutritional conditions. Based on scanning and transmission electron microscopy studies, we hypothesized that the surface-to-volume ratio of cells increases with decreasing nutrient concentrations. The transition from smooth to wrinkled cell surface morphology could be one of the adaptation strategies by which YC6860T maximizes its ability to access available nutrients. To characterize the properties of the wrinkled strain, we performed taxonomic and phylogenetic analyses. 16S rRNA gene sequencing results showed that the strain represented a novel, deep-rooting lineage within the order Rhizobiales with the highest similarity of 94.2% to Pseudorhodoplanes sinuspersici RIPI 110T. Whole genome sequencing was also performed to characterize its genetic features. The strain YC6860T might belong to a new genus, named Rugositalea, and a new species, named Rugositalea oryzae, In addition, taxonomic analysis showed that YC6860T is Gram-negative, aerobic, and rod-shaped with large regular wrinkles resembling a delicate twist of fusilli, measuring 0.5-0.6 {micro}m in width and 1.5-1.6 {micro}m in length under nutrient-limiting conditions. This unique cell structure with regular rugosity could be the first finding that has not been reported in the existing bacterial morphology.

microbiology↗

bioRxiv2022

Lung endothelium instructs dormancy of susceptible metastatic tumour cells

During metastasis, cancer cells hijack blood vessels and travel via the circulation to colonize distant sites1,2. Due to the rarity of these events, the immediate cell fate decisions of arrested circulating tumour cells (aCTC) are poorly understood and the role of the endothelium, as the interface of dissemination, remains elusive3,4. Here, we developed a novel strategy to specifically enrich for aCTC subpopulations capturing all cell states of the extravasation process and, in combination with single cell RNA-sequencing, provide a first blueprint of the transcriptional basis of early aCTC decisions. Upon their arrest at the metastatic site, tumour cells either started proliferating intravascularly or extravasated and preferably reached a state of quiescence. Endothelial-derived angiocrine Wnt factors were found to drive this bifurcation by inducing a mesenchymal-like phenotype in aCTCs instructing them to follow the extravasation-dormancy branch. Surprisingly, homogenous tumour cell pools showed an unexpected baseline heterogeneity in Wnt signalling activity and epithelial-to-mesenchyme-transition (EMT) states. This heterogeneity was established at the epigenetic level and served as the driving force of aCTC behaviour. Hypomethylation enabled high baseline Wnt and EMT activity in tumour cells leading them to preferably follow the extravasation-dormancy route, whereas methylated tumour cells had low activity and proliferated intravascularly. The data identify the pre-determined methylation status of disseminated tumour cells as a key regulator of aCTC behaviour in the metastatic niche. While metastatic niche-derived factors per default instruct the acquisition of quiescence, aCTCs unwind a default proliferation program and only deviate from it if hypomethylation in key gene families renders them responsive towards the microenvironment.

cancer biology↗

bioRxiv2022

Bacterial Infection of the Placenta Induces Sex-Specific Responses in the Fetal Brain

BACKGROUNDEpidemiological data indicate that prenatal infection is associated with an increased risk of several neurodevelopmental disorders in the progeny. These disorders display sex differences in presentation. The role of the placenta, which is a target of prenatal infection, in the sex-specificity of neurodevelopmental abnormalities is unknown. We used an imaging-based animal model of the bacterial pathogen Listeria monocytogenes to identify sex-specific effects of placental infection on neurodevelopment of the fetus. METHODSPregnant CD1 mice were infected with a bioluminescent strain of Listeria on embryonic day 14.5 (E14.5). Excised fetuses were imaged on E18.5 to identify the infected placentas. The associated fetal brains were analyzed for gene expression and altered brain structure due to infection. The behavior of adult offspring affected by prenatal Listeria infection was analyzed. RESULTSPlacental infection induced sex-specific alteration of gene expression patterns in the fetal brain and resulted in abnormal cortical development correlated with placental infection levels. Furthermore, male offspring exhibited abnormal social interaction, whereas females exhibited elevated anxiety. CONCLUSIONPlacental infection by Listeria induced sex-specific abnormalities in neurodevelopment of the fetus. Prenatal infection also affected the behavior of the offspring in a sex-specific manner. ImpactO_LIPlacental infection with Listeria monocytogenes induces sexually dichotomous gene expression patterns in the fetal brain. C_LIO_LIAbnormal cortical lamination is correlated with placental infection levels. C_LIO_LIPlacental infection results in autism related behavior in male offspring and heightened anxiety level in female offspring. C_LI

microbiology↗

bioRxiv2022

Listeria monocytogenes Infection Alters Extracellular Vesicles Produced by Trophoblast Stem Cells to Confer a Pro-Inflammatory State

Placental immunity is critical for fetal health during pregnancy, as invading pathogens can be transmitted from the maternal blood to the fetus through this organ. However, inflammatory responses in the placenta can adversely affect both the fetus and the pregnant mother, and the balance between protective placental immune response and detrimental inflammation is poorly understood. Extracellular vesicles (EVs) are membrane-enclosed vesicles that play a critical role in placental immunity. EVs produced by placental trophoblasts mediate immune tolerance to the fetus and to the placenta itself, but these EVs can also activate detrimental inflammatory responses. The regulation of these effects is not well-characterized, and the role of trophoblast EVs (tEVs) in the response to infection has yet to be defined. The Gram-positive bacterial pathogen Listeria monocytogenes (Lm) infects the placenta, serving as a model to study tEV function in this context. We investigated the effect of Lm infection on the production and function of tEVs, using a trophoblast stem cell (TSCs) model. We found that tEVs from infected TSCs were immunostimulatory, activating macrophages to a pro-inflammatory state. Surprisingly, this activation made RAW 264.7 macrophages more susceptible to subsequent Lm infection. Increased susceptibility to infection has not previously been reported as an effect of EVs. Proteomic analysis and RNA sequencing revealed that tEVs from infected TSCs had altered cargo compared to those from uninfected TSCs. Together, these results suggest an immunomodulatory role for tEVs during prenatal infection.

immunology↗

bioRxiv2022

DeepParcellation: a novel deep learning method for robust brain magnetic resonance imaging parcellation in older East Asians

Accurate parcellation of cortical regions is crucial for distinguishing morphometric changes in aged brains, particularly in degenerative brain diseases. Normal aging and neurodegeneration precipitate brain structural changes, leading to distinct tissue contrast and shape in people aged > 60 years. Manual parcellation by trained radiologists can yield a highly accurate outline of the brain; however, analyzing large datasets is laborious and expensive. Alternatively, newly-developed computational models can quickly and accurately conduct brain parcellation, although thus far only for the brains of Caucasian individuals. DeepParcellation, our novel deep learning model for 3D magnetic resonance imaging (MRI) parcellation, was trained on 5,035 brains of older East Asians (Gwangju Alzheimers & Related Dementia) and 2,535 brains of Caucasians. We trained full 3D models for N-way individual regions of interest using memory reduction techniques. Our method showed the highest similarity and robust reliability among age-ethnicity groups, especially when parcellating the brains of older East Asians.

neuroscience↗

bioRxiv2022

Ultramicrobacteria in various fermented cabbages

Little is known about the ultramicrobial communities of foods. Several bacteria, including pathogenic species, can form ultramicrobial communities when exposed to harsh conditions, making their detection via conventional culture techniques difficult. This study aimed to explore ultramicrobial communities within the microbial communities of fermented cabbage products, such as kimchi, sauerkraut, and suancai, which are produced through specific manufacturing methods based on the laws and culture of respective regions. We used single-molecule real-time sequencing with tangential flow filtration for fermented cabbages after pre-filtration and transmission electron microscopy to confirm the identity of ultramicrobacteria (UMB).To the best of our knowledge, this is the first study to identify the differences between ultramicrobial communities and microbial communities of fermented cabbages. Although the size of the ultramicrobial communities was smaller than that of the latter, their diversity was not lower. In addition, some UMB underwent cell shrinkage due to unfavorable environments, while others maintained their small size regardless. Major pathogens were not detected in the ultramicrobial communities of fermented cabbages. Nevertheless, several suspicious strains were detected. Our method can be used to screen food materials for the presence of UMB undetectable via conventional methods. Ultramicrobial and microbial communities were efficiently separated using tangential flow filtration and analyzed via single-molecule real-time sequencing. The ultramicrobial communities of fermented vegetables were different from conventional microbial communities. This study provides new insights into the ecology of UMB in foods.

microbiology↗

bioRxiv2021

BTBD9 is a novel component of IGF signaling and regulates manganese-induced dopaminergic dysfunction

Restless legs syndrome (RLS) is a common neurological disorder associated with iron deficiency and dopaminergic (DAergic) neuronal dysfunction. BTBD9 is a genetic risk factor for RLS. However, its molecular function remains largely unknown. Here, we report the interaction between BTBD9, manganese (Mn) and insulin/insulin-like growth factor (IGF) signaling in Caenorhabditis elegans, mouse Neuro2a cells and humans. We found that elevated Mn downregulated BTBD9 mRNA levels; in turn, BTBD9 expression attenuated Mn-induced cellular stress and dopaminergic neurodegeneration. As Mn is a known co-factor for insulin receptor and IGF-1 receptor, which activates IGF signaling, we posited that BTBD9 negatively regulates IGF signaling. Our results showed that the protective effects of BTBD9 against Mn toxicity were dependent on the forkhead box O (FOXO) protein. Furthermore, BTBD9 overexpression significantly elevated FOXO level and decreased PKB level, while phosphoinositide-dependent kinase-1 (PDK1) level remained unchanged. We conclude that BTBD9 acts as a key component in the IGF signaling pathway. Meanwhile, the roles of Mn in DAergic neurotoxicity and regulating BTBD9 shed new light on the etiology of RLS.

neuroscience↗

bioRxiv2021

Typical somatomotor physiology of the hand is preserved in a patient with an amputated arm

Electrophysiological signals in the human motor system may change in different ways after deafferentation, with some studies emphasizing reorganization while others propose retained physiology. Understanding whether motor electrophysiology is retained over longer periods of time can be invaluable for patients with paralysis (e.g. ALS or brainstem stroke) when signals from sensorimotor areas may be used for communication or control over neural prosthetic devices. In addition, a maintained electrophysiology can potentially benefit the treatment of phantom limb pains through prolonged use of these signals in a brain-machine interface (BCI). Here, we were presented with the unique opportunity to investigate the physiology of the sensorimotor cortex in a patient with an amputated arm using electrocorticographic (ECoG) measurements. While implanted with an ECoG grid for clinical evaluation of electrical stimulation for phantom limb pain, the patient performed attempted finger movements with the contralateral (lost) hand and executed finger movements with the ipsilateral (healthy) hand. The electrophysiology of the sensorimotor cortex contralateral to the amputated hand remained very similar to that of hand movement in healthy people, with a spatially focused increase of high-frequency band (65-175Hz; HFB) power over the hand region and a distributed decrease in low-frequency band (15-28Hz; LFB) power. The representation of the three different fingers (thumb, index and little) remained intact and HFB patterns could be decoded using support vector learning at single-trial classification accuracies of >90%, based on the first 1-3s of the HFB response. These results demonstrate that hand representations are largely retained in the motor cortex. The intact physiological response of the amputated hand, the high distinguishability of the fingers and fast temporal peak are encouraging for neural prosthetic devices that target the sensorimotor cortex.

neuroscience↗

bioRxiv2020

Efficacy and gut dysbiosis of gentamicin-intercalated smectite as a new therapeutic agent against Helicobacter pylori in a mouse model

BackgroundHelicobacter pylori eradication rate with conventional standard therapy is decreasing owing to antibiotic resistance, necessitating novel antibacterial strategies against H. pylori. We evaluated the efficacy of a gentamicin-intercalated smectite hybrid (S-GM)-based treatment, and analyzed fecal microbiome composition in H. pylori-infected mice. MethodologyTo evaluate anti-H. pylori efficacy, mice were divided into eight groups, and H. pylori eradication was assessed by Campylobacter-like organism (CLO) test and PCR assay of H. pylori in gastric mucosa. One week after H. pylori eradication, proinflammatory cytokine levels and atrophic changes in gastric mucosa were examined. Stool specimens were collected and analyzed for microbiome changes. The S-GM-based triple regimen decreased bacterial burden in vivo, compared with that in untreated mice or mice treated with other regimens. The therapeutic reactions in the CLO test from gastric mucosa were both 90% in standard triple therapy and S-GM therapy group, respectively. Those of H. pylori PCR in mouse gastric mucosa were significantly lower in standard triple therapy and S-GM therapy groups than in non-treatment group. Toxicity test results showed that S-GM therapy reduced IL-8 level and atrophic changes in gastric mucosa. Stool microbiome analysis revealed that compared with mice treated with the standard triple therapy, mice treated with the S-GM therapy showed microbiome diversity and abundant microorganisms at the phylum level. ConclusionOur results suggested that S-GM is a promising and effective therapeutic agent against H. pylori infection. Author summaryThe eradication rate on Helicobacter pylori (H. pylori) showed decreasing trend due to antibiotic resistance, especially clarithromycin. Therefore, we made a smectite hybrid as a drug delivery system using aminoglycosides antibiotic-gentamicin, and applied it to the mouse stomach wall to confirm the localized therapeutic effect, and set the different treatment duration to verify the effect. As a result, it was confirmed that the therapeutic efficacy of gentamicin (GM)-intercalated smectite hybrid (S-GM) was not inferior to the existing standard triple therapy, based on amoxicillin and clarithromycin, and preserved the diversity of gut microbiome composition. Therefore, a S-GM treatment is expected to be a new alternative regimen to H. pylori infection.

microbiology↗

bioRxiv2019

The Effect of New Nurses' Clinical Competence on Career Adaptation

BackgroundNew nurses need a certain period of time to adapt to the organization due to a lack of clinical competence, and also immaturity in social skill. The purpose of this study was to evaluate the effects of new nurses clinical competence on career adaptation in order to use the results as basic information for developing education programs to improve their adaptability in clinical practice. MethodThis study employed a descriptive survey to investigate the clinical competence and the career adaptability of new nurses working in a general hospital. The study subjects were 61 new nurses with less than one year of work experience and data was collected from June, 2018 to July, 2019. Data was analyzed using frequency analysis, descriptive statistics, t-test, ANOVA, Pearsons correlation coefficients, and multiple regression analysis. ResultsOn average, the study subjects have worked for 11.33{+/-}1.51 months. In this study, the average clinical competence of new nurses was 2.21{+/-}0.61, and career adaptability was 3.00{+/-}0.39. The clinical competence of new nurses was positively correlated with career adaptability (r=.453, p<.001). Among the sub-categories of clinical competence, developing professional and legal implementation were found to affect career adaptability (t=2.24, p=.030). ConclusionThe clinical competence of the new nurses was shown to positively affect their career adaptability, and it was confirmed that developing professional and legal implementation in the subcategories of clinical competence affected the career adaptability of the new nurses. Thus, it is necessary to establish a program that provides support for new nurses to enable them to build professional identities that they can be proud of.

scientific communication and education↗

bioRxiv2019

Electrode pooling: How to boost the yield of switchable silicon probes for neuronal recordings

State-of-the-art silicon probes for electrical recording from neurons have thousands of recording sites. However, due to volume limitations there are typically many fewer wires carrying signals off the probe, which restricts the number of channels that can be recorded simultaneously. To overcome this fundamental constraint, we propose a novel method called electrode pooling that uses a single wire to serve many recording sites through a set of controllable switches. Here we present the framework behind this method and an experimental strategy to support it. We then demonstrate its feasibility by implementing electrode pooling on the Neuropixels 1.0 electrode array and characterizing its effect on signal and noise. Finally we use simulations to explore the conditions under which electrode pooling saves wires without compromising the content of the recordings. We make recommendations on the design of future devices to take advantage of this strategy.

neuroscience↗

bioRxiv2019

Vaccination with single plasmid DNA encoding IL-12 and antigens of severe fever with thrombocytopenia syndrome virus elicits complete protection in IFNAR knockout mice

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease caused by SFTS virus (SFTSV) infection. Despite a gradual increase of SFTS cases and high mortality in endemic regions, no specific viral therapy nor vaccine is available. Here, we developed a single recombinant plasmid DNA encoding SFTSV genes, Gn and Gc together with NP-NS fusion antigen, as a vaccine candidate. The viral antigens were fused with Fms-like tyrosine kinase-3 ligand (Flt3L) and IL-12 gene was incorporated into the plasmid to enhance cell-mediated immunity. Vaccination with the DNA provides complete protection of IFNAR KO mice upon lethal SFTSV challenge, whereas immunization with a plasmid without IL-12 gene resulted in partial protection. Since we failed to detect antibodies against surface glycoproteins, Gn and Gc, in the immunized mice, antigen-specific cellular immunity, as confirmed by enhanced antigen-specific T cell responses, might play major role in protection. Finally, we evaluated the degree of protective immunity provided by protein immunization of the individual glycoprotein, Gn or Gc. Although both protein antigens induced a significant level of neutralizing activity against SFTSV, Gn vaccination resulted in relatively higher neutralizing activity and better protection than Gc vaccination. However, both antigens failed to provide complete protection. Given that DNA vaccines have failed to induce sufficient immunogenicity in human trials when compared to protein vaccines, optimal combinations of DNA and protein elements, proper selection of target antigens, and incorporation of efficient adjuvant, need to be further investigated for SFTSV vaccine development.\n\nAuthor summarySevere fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne infection endemic to East Asia including China, Korea, and Japan. Gradual rise of disease incidence and relatively high mortality have become a serious public health problem in the endemic countries. In this study, we developed a recombinant plasmid DNA encoding four antigens, Gn, Gc, NP, and NS, of SFTS virus (SFTSV) as a vaccine candidate. In order to enhance cell-mediated immunity, the viral antigens were fused with Flt3L and IL-2 gene was incorporated into the plasmid. Immunization with the DNA vaccine provides complete protection against lethal SFTSV infection in IFNAR KO mice. Antigen-specific T cell responses might play a major role in the protection since we observed enhanced T cell responses specific to the viral antigens but failed to detect neutralizing antibody in the immunized mice. When we immunized with either viral glycoprotein, Gn protein induced relatively higher neutralizing activity and better protection against SFTSV infection than Gc antigen, but neither generated complete protection. Therefore, an optimal combination of DNA and protein elements, as well as proper selection of target antigens, might be required to produce an effective SFTSV vaccine.

microbiology↗

bioRxiv2019

A ClyA nanopore tweezer for analysis of functional states of protein-ligand interactions

Conformational changes of proteins are essential to their functions. Yet it remains challenging to measure the amplitudes and timescales of protein motions. Here we show that the ClyA nanopore can be used as a molecular tweezer to trap a single maltose-binding protein (MBP) within its lumen, which allows conformation changes to be monitored as electrical current fluctuations in real time. The current measurements revealed three distinct ligand-bound states for MBP in the presence of reducing saccharides. Our biochemical and kinetic analysis reveal that these three states represented MBP bound to different isomers of reducing sugars. These findings shed light on the mechanism of substrate recognition by MBP and illustrate that the nanopore tweezer is a powerful, label-free, single-molecule approach for studying protein conformational dynamics under functional conditions.

biophysics↗

bioRxiv2019

Lattice arrangement of myosin filaments correlates with fiber type in rat skeletal muscle

The thick (myosin-containing) filaments of vertebrate skeletal muscle are arranged in a hexagonal lattice, interleaved with an array of thin (actin-containing) filaments with which they interact to produce contraction. X-ray diffraction and electron microscopy have shown that there are two types of thick filament lattice. In the simple lattice, all filaments have the same orientation about their long axis, while in the super lattice, nearest neighbors have rotations differing by 60{degrees}. Tetrapods (amphibians, reptiles, birds, mammals) typically have only a super lattice, while the simple lattice is confined to fish. We have carried out X-ray diffraction and electron microscopy of the soleus (SOL) and extensor digitorum longus (EDL) muscles of the rat and found that while the EDL has a super-lattice, as expected, the SOL has a simple lattice. The EDL and SOL of the rat are unusual in being essentially pure fast and slow muscles respectively. The mixed fiber content of most tetrapod muscles and/or lattice disorder may explain why the simple lattice has not been apparent in these vertebrates before. This is supported by only weak simple lattice diffraction in the X-ray pattern of mouse SOL, which has a greater mix of fiber types than rat. We conclude that the simple lattice might be common in tetrapods. The correlation between fiber type and filament lattice arrangement suggests that the lattice arrangement may contribute to the functional properties of a muscle.\n\nSummaryThe three-dimensional arrangement of thick filaments in skeletal muscle is studied by X-ray diffraction and electron microscopy. A correlation is found between thick filament lattice type (simple or super lattice) and fiber type (fast/slow). This suggests that lattice organization contributes to muscle functional properties

biophysics↗

bioRxiv2019

A stealth adhesion factor contributes to Vibrio vulnificus pathogenicity: Flp pili play roles in host invasion, survival in the blood stream and resistance to complement activation

The tad operons encode the machinery required for adhesive Flp (fimbrial low-molecular-weight protein) pili biogenesis. Vibrio vulnificus, an opportunistic pathogen, harbors three distinct tad loci. Among them, only tad1 locus was highly upregulated in in vivo growing bacteria compared to in vitro culture condition. To understand the pathogenic roles of the three tad loci during infection, we constructed single, double and triple tad loci deletion mutants. Interestingly, only the{Delta} tad123 triple mutant cells exhibited significantly decreased lethality in mice. Ultrastructural observations revealed short, thin filamentous projections disappeared on the{Delta} tad123 mutant cells. Since the pilin was paradoxically non-immunogenic, a V5 tag was fused to Flp to visualize the pilin protein by using immunogold EM and immunofluorescence microscopy. The{Delta} tad123 mutant cells showed attenuated host cell adhesion, delayed RtxA1 exotoxin secretion and subsequently impaired translocation across the intestinal epithelium compared to wild type, which could be partially complemented with each wild type operon. The{Delta} tad123 mutant was susceptible to complement-mediated bacteriolysis, predominantly via the alternative pathway, suggesting stealth hiding role of the Tad pili. Taken together, all three tad loci cooperate to confer successful invasion of V. vulnificus into deeper tissue and evasion from host defense mechanisms, ultimately resulting in septicemia.\n\nAuthor SummaryTo understand the roles of the three Tad operons in the pathogenesis of V. vulnificus infection, we constructed mutant strain with single, double and triple Tad loci deletions. Employing a variety of mouse infection models coupled with molecular genetic analyses, we demonstrate here that all three Tad operons are required for V. vulnificus pathogenicity as the cryptic pili contribute to host cell and tissue invasion, survival in the blood, and resistance to complement activation.

microbiology↗

bioRxiv2019

The central role of the tail in switching off myosin II in cells

Myosin II is a motor protein playing an essential role in cell motility. The molecule can exist as a polymer that pulls on actin to generate motion, or as an inactive monomer with a compact structure, in which its tail is folded and its two heads interact with each other. This conformation functions in cells as an energy-conserving storage and transport molecule. The mechanism of inhibition is not fully understood. We have carried out a 3D reconstruction of the switched-off form revealing for the first time multiple interactions between the tail and the two heads that trap ATP hydrolysis products, block actin binding, obstruct head phosphorylation, and prevent filament formation. Blocking these essential features of myosin function can explain the high degree of inhibition of the folded form of myosin, serving its energy-conserving, storage function in cells. The structure also suggests a mechanism for unfolding when activated by phosphorylation.

cell biology↗