Development and web deployment of prediction model for pulmonary arterial pressure in chronic thromboembolic pulmonary hypertension using machine learning.

BACKGROUND AND PURPOSE: Mean pulmonary artery pressure (mPAP) is a key index for chronic thromboembolic pulmonary hypertension (CTEPH). Using machine learning, we attempted to construct an accurate prediction model for mPAP in patients with CTEPH. METHODS: A total of 136 patients diagnosed with CTEPH were included, for whom mPAP was measured. The following patient data were used as explanatory variables in the model: basic patient information (age and sex), blood tests (brain natriuretic peptide (BNP)), echocardiography (tricuspid valve pressure gradient (TRPG)), and chest radiography (cardiothoracic ratio (CTR), right second arc ratio, and presence of avascular area). Seven machine learning methods including linear regression were used for the multivariable prediction models. Additionally, prediction models were constructed using the AutoML software. Among the 136 patients, 2/3 and 1/3 were used as training and validation sets, respectively. The average of R squared was obtained from 10 different data splittings of the training and validation sets. RESULTS: The optimal machine learning model was linear regression (averaged R squared, 0.360). The optimal combination of explanatory variables with linear regression was age, BNP level, TRPG level, and CTR (averaged R squared, 0.388). The R squared of the optimal multivariable linear regression model was higher than that of the univariable linear regression model with only TRPG. CONCLUSION: We constructed a more accurate prediction model for mPAP in patients with CTEPH than a model of TRPG only. The prediction performance of our model was improved by selecting the optimal machine learning method and combination of explanatory variables.

Plasmonic Manipulation of DNA using a Combination of Optical and Thermophoretic Forces: Separation of Different-Sized DNA from Mixture Solution.

We demonstrate the size-dependent separation and permanent immobilization of DNA on plasmonic substrates by means of plasmonic optical tweezers. We found that a gold nanopyramidal dimer array enhanced the optical force exerted on the DNA, leading to permanent immobilization of the DNA on the plasmonic substrate. The immobilization was realized by a combination of the plasmon-enhanced optical force and the thermophoretic force induced by a photothermal effect of the plasmons. In this study, we applied this phenomenon to the separation and fixation of size-different DNA. During plasmon excitation, DNA strands of different sizes became permanently immobilized on the plasmonic substrate forming micro-rings of DNA. The diameter of the ring was larger for longer DNA (in base pairs). When we used plasmonic optical tweezers to trap DNA of two different lengths dissolved in solution (φx DNA (5.4 kbp) and λ-DNA (48.5 kbp), or φx DNA and T4 DNA (166 kbp)), the DNA were immobilized, creating a double micro-ring pattern. The DNA were optically separated and immobilized in the double ring, with the shorter sized DNA and the larger one forming the smaller and larger rings, respectively. This phenomenon can be quantitatively explained as being due to a combination of the plasmon-enhanced optical force and the thermophoretic force. Our plasmonic optical tweezers open up a new avenue for the separation and immobilization of DNA, foreshadowing the emergence of optical separation and fixation of biomolecules such as proteins and other ncuelic acids.

Plasmon-Induced Selective Oxidation Reaction at Single-Walled Carbon Nanotubes.

Local surface plasmon resonance (LSPR)-induced oxidation of semiconducting and metallic single-walled nanotubes (SWNTs) on the nanometer scale was investigated using surface-enhanced Raman scattering (SERS) measurements. An isolated SWNT was supported on a well-defined Au nanodimer structure that possesses an LSPR field at the nanogap under light irradiation, and highly intense SERS spectra of the SWNT at the gap region were measured. SERS analysis under O2-saturated solutions and the addition of reactive oxygen species inhibitors demonstrated that condensed singlet oxygen (1O2), which is one of the reactive oxygen species, was efficiently generated from a semiconducting SWNT at the nanogap by the LSPR field and led to the local oxidation of the tube. In contrast to the semiconducting SWNT, no defect formation was observed in a metallic SWNT, probably because of rapid quenching of the photoexcited state. This selective local defect formation by LSPR-induced oxidation of a semiconducting SWNT would provide novel nanoprocessing and nanofunctionalization methods for the fabrication of future SWNT-based nanodevices.

Pharmacogenetic reactivation of the original engram evokes an extinguished fear memory.

Fear memory extinction has several characteristic behavioral features, such as spontaneous recovery, renewal, and reinstatement, suggesting that extinction training does not erase the original association between the conditioned stimulus (CS) and the unconditioned stimulus (US). However, it is unclear whether reactivation of the original physical record of memory (i.e., memory trace) is sufficient to produce conditioned fear response after extinction. Here, we performed pharmacogenetic neuronal activation using transgenic mice expressing hM3Dq DREADD (designer receptor exclusively activated by designer drug) under the control of the activity-dependent c-fos gene promoter. Neuronal ensembles activated during fear-conditioned learning were tagged with hM3Dq and subsequently reactivated after extinction training. The mice exhibited significant freezing, even when the fear memory was no longer triggered by external CS, indicating that the artificial reactivation of a specific neuronal ensemble was sufficient to evoke the extinguished fear response. This freezing was not observed in non-fear-conditioned mice expressing hM3dq in the same brain areas. These results directly demonstrated that at least part of the original fear memory trace remains after extinction, and such residual plasticity might reflect the persistent memory.

Involvement of two-component signal transduction system, ComDE, in the regulation of growth and genetic transformation, in the ruminal bacterium Streptococcus bovis.

In ruminants, Streptococcus bovis is considered to be associated with acute rumen acidosis. To elucidate the regulatory mechanisms of S. bovis growth, we investigated the function of the two components of the peptide pheromone-signaling system, ComD and ComE, which are encoded by comD and comE, respectively, via the competence-stimulating peptide ComC, which is encoded by comC. Deletion of entire comC and two-thirds of comD resulted in decreased growth rate, which may be related to the change in the expression of several proteins, as shown by two-dimensional gel electrophoresis. The transcript level of comED was decreased by the disruption of comCD, suggesting that the transcription of comED might be stimulated by ComC. The transformation frequency was decreased by the disruption of comCD. Addition of recombinant ComC to S. bovis cultures increased the growth rate and transformation frequency. In the cultures of mixed ruminal microbes, addition of mature ComC peptide increased the number of S. bovis per total bacterial counts as estimated by the cDNA amounts of 16SrRNA. Thus, the peptide pheromone-signaling system via ComC, D, and E might be involved in the control of S. bovis growth in addition to competence development. This is the first report suggesting that an autoinducing peptide functions in the ruminal ecosystem.

Characterization and transcription of the genes involved in butyrate production in Butyrivibrio fibrisolvens type I and II strains.

The genes in Butyrivibrio fibrisolvens that encode the enzymes involved in butyrate production were sequenced. In a type I strain (ATCC 19171(T)), the genes coding for the enzymes that catalyze the conversion from acetyl-CoA to butyryl-CoA, thl (thiolase), crt (crotonase), hbd (beta-hydroxybutyryl-CoA dehydrogenase), bcd (butyryl-CoA dehydrogenase), etfB (electron transfer flavoprotein [ETF]-beta), and etfA (ETF-alpha), were found to be clustered and arranged in this order. A type II strain (ATCC 51255) had the same clustered genes with the same arrangement, except that crt was not present in the clustered genes. The deduced amino acid sequences of these enzymes did not greatly differ between the two strains, and even between B. fibrisolvens and clostridia. Amino acid identity appeared to be higher within the same type than between types I and II. The clustered genes were shown to be cotranscribed, and constitutively transcribed without being affected significantly by culture conditions.

Molecular characterization and transcription of the luxS gene that encodes LuxS autoinducer 2 synthase in Streptococcus bovis.

Presence of the luxS gene that encodes LuxS autoinducer 2 (AI-2) synthase in Streptococcus bovis was demonstrated, and the molecular properties and transcription of the gene were examined. The S. bovis luxS was transcribed in a monocistronic fashion. Intracellular luxS-mRNA increased sharply during the initial exponential growth, and decreased abruptly after the middle exponential phase. The large drop in luxS transcription began before the glucose supply to cells decreased or the growth rate declined. Transcription of luxS was not directly related to cell density, and continued at a maximal rate when cells were kept growing at a maximal rate. It is conceivable that AI-2 activity in S. bovis acts as a signal for adjusting cell physiology and metabolism in response to environmental conditions. However, the role of LuxS in S. bovis, including the regulation of AI-2 synthesis,, remains to be clarified.

Molecular characterization of CcpA and involvement of this protein in transcriptional regulation of lactate dehydrogenase and pyruvate formate-lyase in the ruminal bacterium Streptococcus bovis.

A ccpA gene that encodes global catabolite control protein A (CcpA) in Streptococcus bovis was identified and characterized, and the involvement of CcpA in transcriptional control of a gene (ldh) encoding lactate dehydrogenase (LDH) and a gene (pfl) encoding pyruvate formate-lyase (PFL) was examined. The ccpA gene was shown to be transcribed as a monocistronic operon. A catabolite-responsive element (cre) was found in the promoter region of ccpA, suggesting that ccpA transcription in S. bovis is autogenously regulated. CcpA required HPr that was phosphorylated at the serine residue at position 46 (HPr-[Ser-P]) for binding to the cre site, but glucose 6-phosphate, fructose 1,6-bisphosphate, and NADP had no effect on binding. Diauxic growth was observed when S. bovis was grown in a medium containing glucose and lactose, but it disappeared when ccpA was disrupted, which indicates that CcpA is involved in catabolite repression in S. bovis. The level of ccpA mRNA was higher when cells were grown on glucose than when they were grown on lactose, which was in line with the level of ldh mRNA. When cells were grown on glucose, the ldh mRNA level was lower but the pfl mRNA level was higher in a ccpA-disrupted mutant than in the parent strain, which suggests that ldh transcription is enhanced and pfl transcription is suppressed by CcpA. The ccpA-disrupted mutant produced less lactate and more formate than the parent, probably because the mutant had reduced LDH activity and elevated PFL activity. In the upper region of both ldh and pfl, a cre-like sequence was found, suggesting that the complex consisting of CcpA and HPr-[Ser-P] binds to the possible cre sites. Thus, CcpA appears to be involved in the global regulation of sugar utilization in S. bovis.

Molecular characteristics of phosphoenolpyruvate: mannose phosphotransferase system in Streptococcus bovis.

To elucidate the regulatory mechanism of catabolite control in Streptococcus bovis, we investigated the molecular properties and gene expression of the mannose-specific phosphoenolpyruvate (PEP)-dependent sugar: phosphotransferase system (PTS). The mannose PTS gene cluster (man) was found to comprise a gene encoding enzyme (E) II AB (manL) and genes encoding EIIC (manM), EIID (manN), and a putative regulator (manO). The gene cluster (man operon) was transcribed from one transcriptional start site, which was located 40 bp upstream of the manL start codon. However, two transcriptional start sites were found between manN and manO in primer extension analysis, and the manO may be transcribed independently from the man operon. The man operon and manO were constitutively transcribed without being affected by culture conditions, such as the sugar supplied (glucose, galactose, fructose, maltose, lactose, sucrose, or mannose), growth rate, or pH.

Molecular characterization and transcriptional regulation of nitrate reductase in a ruminal bacterium, Selenomonas ruminantium.

Nitrate reductase (NaR) of a strain of Selenomonas ruminantium was purified, and the gene encoding NaR (nar) was sequenced. The 6.4 kbp nar gene consisted of narG, H, J, and I in this order. The deduced amino acid sequences of these subunits resembled those of membrane-bound nitrate reductase-A reported for Escherichia coli. It was shown that narG, H, J, and I are transcribed as a single polycistronic message (nar operon). The level of intracellular nar-mRNA was higher when S. ruminantium was grown with nitrate than when grown without nitrate, suggesting that nar transcription is enhanced by nitrate. The level of nar-mRNA, which was in parallel to the amount of NaR per cellular nitrogen, was suggested to be enhanced in response to the deficiency of energy and electron supply. Therefore, NaR synthesis in S. ruminantium appeared to be regulated at the transcriptional level in response to the availability of energy and electrons. S. ruminantium reduced nitrate and fumarate simultaneously with no significant effect of fumarate on nar transcription. Addition of fumarate stimulated nitrate reduction, which was caused by increased cell growth because of increased acquirement of ATP via electron transport phosphorylation coupled with fumarate reduction.

Effects of the overexpression of fructose-1,6-bisphosphate aldolase on fermentation pattern and transcription of the genes encoding lactate dehydrogenase and pyruvate formate-lyase in a ruminal bacterium, Streptococcus bovis.

Whether fructose-1,6-bisphosphate (FBP) triggers the transcriptional regulation of the gene expression of lactate dehydrogenase (LDH) and pyruvate formate-lyase (PFL) in Streptococcus bovis was examined by constructing a recombinant strain that overexpresses FBP aldolase (FBA). When the recombinant strain was grown on glucose, intracellular FBP was much lower as compared to the parent strain, whereas dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde-3-phosphate (GAP) were slightly higher. Intracellular ATP and ADP were slightly lower, but the NADH/NAD(+) ratio was not different. When glucose was replaced by lactose, a less readily utilized substrate, there was no great difference in FBP, DHAP, GAP, or adenine nucleotides. Overexpression of FBA decreased the level of LDH-mRNA, and increased the level of PFL-mRNA. Consequently, FBP concentration was positively related to the LDH-mRNA level and inversely related to the PFL-mRNA level. On the contrary, DHAP and GAP concentrations were positively related to the PFL-mRNA level and inversely related to the LDH-mRNA level. The levels of these mRNA were proportional to the amounts of corresponding enzymes in cells. As a result, the ratio of formate to lactate produced was increased by the overexpression of FBA. From these results, it could be presumed that FBP is involved in the transcriptional control of LDH and PFL synthesis in S. bovis.

Molecular characteristics and transcription of the gene encoding a multifunctional alcohol dehydrogenase in relation to the deactivation of pyruvate formate-lyase in the ruminal bacterium Streptococcus bovis.

To clarify the deactivation mechanism of pyruvate formate-lyase (PFL) and its role in the regulation of fermentation in Streptococcus bovis, the molecular properties and genetic expression of multifunctional alcohol dehydrogenase (ADHE) were investigated. S. bovis was found to have ADHE, which was deduced to consist of 872 amino acids with a molecular mass of 97.4 kDa. The ADHE was shown to harbor three enzyme activities: (1) alcohol dehydrogenase, (2) coenzyme-A-linked acetaldehyde dehydrogenase that catalyzes the conversion of acetyl-CoA to ethanol, and (3) PFL deactivase. Similar to Escherichia coli ADHE, S. bovis ADHE required Fe2+ for its activity. The gene encoding ADHE ( adhE) was shown to be monocistronic. The level of adhE mRNA changed in parallel with the mRNA levels of the genes encoding PFL (pfl) and PFL-activating enzyme (act) as the growth conditions changed, although these genes are independently transcribed. Synthesis of ADHE, PFL-activating enzyme, and PFL appears to be regulated concomitantly. Overexpression of ADHE did not cause a change in the formate-to-lactate ratio. It is conceivable that ADHE is not significantly involved in the reversible inactivation of active PFL under anoxic conditions. Partition of the flow from pyruvate appears to be mainly regulated by the activities of lactate dehydrogenase and PFL.