Structures illustrate step-by-step mitochondrial transcription initiation.

Transcription initiation is a key regulatory step in gene expression during which RNA polymerase (RNAP) initiates RNA synthesis de novo, and the synthesized RNA at a specific length triggers the transition to the elongation phase. Mitochondria recruit a single-subunit RNAP and one or two auxiliary factors to initiate transcription. Previous studies have revealed the molecular architectures of yeast1 and human2 mitochondrial RNAP initiation complexes (ICs). Here we provide a comprehensive, stepwise mechanism of transcription initiation by solving high-resolution cryogenic electron microscopy (cryo-EM) structures of yeast mitochondrial RNAP and the transcription factor Mtf1 catalysing two- to eight-nucleotide RNA synthesis at single-nucleotide addition steps. The growing RNA-DNA is accommodated in the polymerase cleft by template scrunching and non-template reorganization, creating stressed intermediates. During early initiation, non-template strand scrunching and unscrunching destabilize the short two- and three-nucleotide RNAs, triggering abortive synthesis. Subsequently, the non-template reorganizes into a base-stacked staircase-like structure supporting processive five- to eight-nucleotide RNA synthesis. The expanded non-template staircase and highly scrunched template in IC8 destabilize the promoter interactions with Mtf1 to facilitate initiation bubble collapse and promoter escape for the transition from initiation to the elongation complex (EC). The series of transcription initiation steps, each guided by the interplay of multiple structural components, reveal a finely tuned mechanism for potential regulatory control.

Cryo-EM Structures Reveal Transcription Initiation Steps by Yeast Mitochondrial RNA Polymerase.

Mitochondrial RNA polymerase (mtRNAP) is crucial in cellular energy production, yet understanding of mitochondrial DNA transcription initiation lags that of bacterial and nuclear DNA transcription. We report structures of two transcription initiation intermediate states of yeast mtRNAP that explain promoter melting, template alignment, DNA scrunching, abortive synthesis, and transition into elongation. In the partially melted initiation complex (PmIC), transcription factor MTF1 makes base-specific interactions with flipped non-template (NT) nucleotides "AAGT" at -4 to -1 positions of the DNA promoter. In the initiation complex (IC), the template in the expanded 7-mer bubble positions the RNA and NTP analog UTPαS, while NT scrunches into an NT loop. The scrunched NT loop is stabilized by the centrally positioned MTF1 C-tail. The IC and PmIC states coexist in solution, revealing a dynamic equilibrium between two functional states. Frequent scrunching/unscruching transitions and the imminent steric clashes of the inflating NT loop and growing RNA:DNA with the C-tail explain abortive synthesis and transition into elongation.

A network medicine approach to investigation and population-based validation of disease manifestations and drug repurposing for COVID-19.

The global coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to unprecedented social and economic consequences. The risk of morbidity and mortality due to COVID-19 increases dramatically in the presence of coexisting medical conditions, while the underlying mechanisms remain unclear. Furthermore, there are no approved therapies for COVID-19. This study aims to identify SARS-CoV-2 pathogenesis, disease manifestations, and COVID-19 therapies using network medicine methodologies along with clinical and multi-omics observations. We incorporate SARS-CoV-2 virus-host protein-protein interactions, transcriptomics, and proteomics into the human interactome. Network proximity measurement revealed underlying pathogenesis for broad COVID-19-associated disease manifestations. Analyses of single-cell RNA sequencing data show that co-expression of ACE2 and TMPRSS2 is elevated in absorptive enterocytes from the inflamed ileal tissues of Crohn disease patients compared to uninflamed tissues, revealing shared pathobiology between COVID-19 and inflammatory bowel disease. Integrative analyses of metabolomics and transcriptomics (bulk and single-cell) data from asthma patients indicate that COVID-19 shares an intermediate inflammatory molecular profile with asthma (including IRAK3 and ADRB2). To prioritize potential treatments, we combined network-based prediction and a propensity score (PS) matching observational study of 26,779 individuals from a COVID-19 registry. We identified that melatonin usage (odds ratio [OR] = 0.72, 95% CI 0.56-0.91) is significantly associated with a 28% reduced likelihood of a positive laboratory test result for SARS-CoV-2 confirmed by reverse transcription-polymerase chain reaction assay. Using a PS matching user active comparator design, we determined that melatonin usage was associated with a reduced likelihood of SARS-CoV-2 positive test result compared to use of angiotensin II receptor blockers (OR = 0.70, 95% CI 0.54-0.92) or angiotensin-converting enzyme inhibitors (OR = 0.69, 95% CI 0.52-0.90). Importantly, melatonin usage (OR = 0.48, 95% CI 0.31-0.75) is associated with a 52% reduced likelihood of a positive laboratory test result for SARS-CoV-2 in African Americans after adjusting for age, sex, race, smoking history, and various disease comorbidities using PS matching. In summary, this study presents an integrative network medicine platform for predicting disease manifestations associated with COVID-19 and identifying melatonin for potential prevention and treatment of COVID-19.

Intestinal obstruction in pregnancy-a rare presentation of uterine perforation.

BACKGROUND: Intestinal obstruction is an uncommon non-obstetric condition during pregnancy which may cause maternal and fetal mortality. Clinicians are confronted with challenges in diagnosis and treatment of intestinal obstruction due to the overlapping symptoms, concerns over radiological evaluation, and surgical risks. CASE PRESENTATION: We reported a 39-year old, gravida 7, para 2, woman who suffered from acute intestinal obstruction at 34 weeks of gestation. Ultrasonography and abdominal computed tomography were applied for intestinal obstruction diagnose. Conservative treatment was initially attempted. But following ultrasound found the absence of fluid in the amniotic sac and the patient showed no improvement in clinical symptoms. An emergency caesarean section was then performed. Intra-operative assessment showed dense adhesion between the left wall of uterus and omentum, descending colon, and sigmoid colon. After adhesion dialysis, uterine rupture with complete opening of the uterine wall at the site of left uterine cornua was found without active bleeding. The uterine rupture was then repaired. CONCLUSIONS: Although uncommon during pregnancy, clinical suspicion of bowel obstruction is necessary especially in women with a history of abdominal surgery. Surgical intervention is indicated when conservative therapy fails and when there are signs of abnormal fetal conditions and worsened symptoms.

Spatial Transcriptomics of Aging Rat Ovaries Reveals Unexplored Cell Subpopulations with Reduced Antioxidative Defense.

INTRODUCTION: Ovarian aging is characterized by a gradual decline in quantity and quality of oocytes and lower chance of fertility. Better understanding the genetic modulation during ovarian aging can further address available treatment options for aging-related ovarian diseases and fertility preservation. METHODS: A novel technique spatial transcriptomics (ST) was used to investigate the spatial transcriptome features of rat ovaries. Transcriptomes from ST spots in the young and aged ovaries were clustered using differentially expressed genes. These data were analyzed to determine the spatial organization of age-induced heterogeneity and potential mechanisms underlying ovarian aging. RESULTS: In this study, ST technology was applied to profile the comprehensive spatial imaging in young and aged rat ovary. Fifteen ovarian cell clusters with distinct gene-expression signatures were identified. The gene expression dynamics of granulosa cell clusters revealed three sub-types with sequential developmental stages. Aged ovary showed a significant decrease in the number of granulosa cells from the antral follicle. Besides, a remarkable rearrangement of interstitial gland cells was detected in aging ovary. Further analysis of aging-associated transcriptional changes revealed that the disturbance of oxidative pathway was a crucial factor in ovarian aging. CONCLUSIONS: This study firstly described an aging-related spatial transcriptome changes in ovary and identified the potential targets for prevention of ovarian aging. These data may provide the basis for further investigations of the diagnosis and treatment of aging-related ovarian disorders.

Effect of Flowering Stages on the Content of Active Ingredients and Antioxidant Capability of Bletilla striata Flowers.

Bletilla striata (Thunb.) Reichb.f. is a perennial herb with abundant active ingredients. Previous research mainly focused on its tubers, however, the study on flowers, especially the variation of active ingredient contents at different flowering stages, was rarely seen. This study analyzed the total phenols, flavonoids, polysaccharides, anthocyanins, and cyanidin-3-O-glucoside content of B. striata flowers which were in cultivated in Herb Garden of Zhejiang A&F University and collected in May, 2019, in order to investigate the changes in active ingredients and antioxidant capacity among different flowering stages (bud, initial, and full bloom). Changes in radical scavenging capability of DPPH (1,1-Diphenyl-2-picrylhydrazyl radical), ABTS (2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonate)), and hydroxy were analyzed. Significant differences in active ingredient content of flowers were detected among different flowering stages. The total phenolic content increased continuously during the entire flowering stage. The contents of total flavonoid, total polysaccharide, and cyanidin-3-O-glucoside reached peaks at the initial blooming stage and then fell as the flowering process continued. The antioxidant activity in initial stage was the highest than in any other flowering stages. Therefore, we conclude that the initial blooming stage is the best harvesting stage of B. striata flowers. This study provides a robust basis for the harvest and utilization of B. striata flowers in food, medical, and cosmetic industries.

Physician-Modified Endograft With Triple Inner Branches for Extensive Aortic Arch Aneurysm.

PURPOSE: The purpose of this case report was to demonstrate the feasibility of a physician-modified endograft (PMEG) with 3 inner branches for extensive aortic arch aneurysm. CASE REPORT: A 69-year-old male presented with extensive aortic arch aneurysm involving all supra-aortic vessels. An Ankura thoracic stent graft was modified with 3 inner branches fashioned of Viabahn endoprostheses. The procedure was technically successful, and the patient was discharged with no complications. CONCLUSION: This back-table modification of the off-the-shelf endograft is an especially attractive option for complex arch pathologies with urgency and deemed too high risk for reopen surgery.

The C-terminal tail of the yeast mitochondrial transcription factor Mtf1 coordinates template strand alignment, DNA scrunching and timely transition into elongation.

Mitochondrial RNA polymerases depend on initiation factors, such as TFB2M in humans and Mtf1 in yeast Saccharomyces cerevisiae, for promoter-specific transcription. These factors drive the melting of promoter DNA, but how they support RNA priming and growth was not understood. We show that the flexible C-terminal tails of Mtf1 and TFB2M play a crucial role in RNA priming by aiding template strand alignment in the active site for high-affinity binding of the initiating nucleotides. Using single-molecule fluorescence approaches, we show that the Mtf1 C-tail promotes RNA growth during initiation by stabilizing the scrunched DNA conformation. Additionally, due to its location in the path of the nascent RNA, the C-tail of Mtf1 serves as a sensor of the RNA-DNA hybrid length. Initially, steric clashes of the Mtf1 C-tail with short RNA-DNA hybrids cause abortive synthesis but clashes with longer RNA-DNA trigger conformational changes for the timely release of the promoter DNA to commence the transition into elongation. The remarkable similarities in the functions of the C-tail and σ3.2 finger of the bacterial factor suggest mechanistic convergence of a flexible element in the transcription initiation factor that engages the DNA template for RNA priming and growth and disengages when needed to generate the elongation complex.

The C-terminal tails of the mitochondrial transcription factors Mtf1 and TFB2M are part of an autoinhibitory mechanism that regulates DNA binding.

The structurally homologous Mtf1 and TFB2M proteins serve as transcription initiation factors of mitochondrial RNA polymerases in Saccharomyces cerevisiae and humans, respectively. These transcription factors directly interact with the nontemplate strand of the transcription bubble to drive promoter melting. Given the key roles of Mtf1 and TFB2M in promoter-specific transcription initiation, it can be expected that the DNA binding activity of the mitochondrial transcription factors is regulated to prevent DNA binding at inappropriate times. However, little information is available on how mitochondrial DNA transcription is regulated. While studying C-terminal (C-tail) deletion mutants of Mtf1 and TFB2M, we stumbled upon a finding that suggested that the flexible C-tail region of these factors autoregulates their DNA binding activity. Quantitative DNA binding studies with fluorescence anisotropy-based titrations revealed that Mtf1 with an intact C-tail has no affinity for DNA but deletion of the C-tail greatly increases Mtf1's DNA binding affinity. Similar observations were made with TFB2M, although autoinhibition by the C-tail of TFB2M was not as complete as in Mtf1. Analysis of available TFB2M structures disclosed that the C-tail engages in intramolecular interactions with the DNA binding groove in the free factor, which, we propose, inhibits its DNA binding activity. Further experiments showed that RNA polymerase relieves this autoinhibition by interacting with the C-tail and engaging it in complex formation. In conclusion, our biochemical and structural analyses reveal autoinhibitory and activation mechanisms of mitochondrial transcription factors that regulate their DNA binding activities and aid in specific assembly of transcription initiation complexes.

Phosphorylation of mitochondrial transcription factor B2 controls mitochondrial DNA binding and transcription.

Mammalian cells contain genetic information in two compartments, the nucleus and the mitochondria. Mitochondrial gene expression must be coordinated with nuclear gene expression to respond to cellular energetic needs. To gain insight into the coordination between the nucleus and mitochondria, there is a need to understand the regulation of transcription of mitochondrial DNA (mtDNA). Reversible protein post-translational modifications of the mtDNA transcriptional machinery may be one way to control mtDNA transcription. Here we focus on a member of the mtDNA transcription initiation complex, mitochondrial transcription factor B2 (TFB2M). TFB2M melts mtDNA at the promoter to allow the RNA polymerase (POLRMT) to access the DNA template and initiate transcription. Three phosphorylation sites have been previously identified on TFB2M by mass spectrometry: threonine 184, serine 197, and threonine 313. Phosphomimetics were established at these positions. Proteins were purified and analyzed for their ability to bind mtDNA and initiate transcription in vitro. Our results indicate phosphorylation at threonine 184 and threonine 313 impairs promoter binding and prevents transcription. These findings provide a potential regulatory mechanism of mtDNA transcription and help clarify the importance of protein post-translational modifications in mitochondrial function.

In-situ recurrence of the primary cardiac dedifferentiated liposarcoma: To resect or not?

Dedifferentiated liposarcoma (DDLPS) is a rare type of neoplasm which can originally rise in cardiac chamber. Owing to the recurrence and distant metastasis, the prognosis of the primary malignant cardiac tumor is extremely poor and remains a challenge for cardiac surgeons. Here, we report a patient with an intracavitary mass which was diagnosed as DDLPS by postoperative pathological examinations, experienced repeated in-situ recurrence of the malignant cardiac tumor.

[The Screening for Long Non-coding RNAs Related to Vein Graft Failure in Rat External Jugular Vein-Carotid Bypass Models].

OBJECTIVE: This study was designed to show the time-dependent changes in the expression profiles of long non-coding RNAs (lncRNAs) in rat vein grafts by using the high-throughput sequencing technique, and subsequently figure out lncRNAs related to vein graft failure. METHODS: The whole SD rats were randomly classified into four groups according to different sampling time points (0, 7, 14 and 28 days after surgery, respectively; n=3 per group). The day 0 group was set as a control, and the other three groups were set as experimental groups (the model of external jugular vein-carotid artery bypass was prepared in the experimental group). We identified the differentially expressed lncRNAs of the vein graft sample at different sampling time points with high-throughput sequencing, and verified these results using the technique of real-time quantitative PCR (RT-qPCR). Meanwhile, we used Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to screen lncRNAs which may play roles in the restenosis process of vein grafts. The function of lncRNA-mRNA pairs was predicted. We subsequently used RT-qPCR to verify these lncRNAs. RESULTS: A total of 2 572 sustained differentially expressed lncRNAs were identified in our study. We showed the top ten differentially expressed lncRNAs at each post-operative time point. Through GO analysis and KEGG pathway analysis, we revealed the sustained differentially expressed genes which may be involved in VGF-related biological process, cellular component, molecular function and biological pathways. Finally, we screened 15 pairs of lncRNA-mRNA, including MRAK083052- Nrp1, which may play roles in mediating the restenosis of vein graft. And the results of RT-qPCR were consistent with the results of the high-throughput sequencing. CONCLUSION: The present study investigated the time-dependent changes in the expression profiles of lncRNAs in vein grafts. We also screened 15 pairs of lncRNA-mRNA which may paly roles in vein graft failure.

PPR-SMR1 is required for the splicing of multiple mitochondrial introns, interacts with Zm-mCSF1, and is essential for seed development in maize.

Group II introns are ribozymes that can excise themselves from precursor-RNA transcripts, but plant organellar group II introns have structural deviations that inhibit ribozyme activity. Therefore, splicing of these introns requires the assistance of nuclear- and/or organellar-encoded splicing factors; however, how these splicing factors function remains unclear. In this study, we report the functions and interactions of two splicing factors, PPR-SMR1 and Zm-mCSF1, in intron splicing in maize mitochondria. PPR-SMR1 is a SMR domain-containing pentatricopeptide repeat (PPR) protein and Zm-mCSF1 is a CRM domain-containing protein, and both are targeted to mitochondria. Loss-of-function mutations in each of them severely arrests embryogenesis and endosperm development in maize. Functional analyses indicate that PPR-SMR1 and Zm-mCSF1 are required for the splicing of most mitochondrial group II introns. Among them, nad2-intron 2 and 3, and nad5-intron 1 are PPR-SMR1/Zm-mCSF1-dependent introns. Protein interaction assays suggest that PPR-SMR1 can interact with Zm-mCSF1 through its N-terminus, and that Zm-mCSF1 is self-interacting. Our findings suggest that PPR-SMR1, a novel splicing factor, acts in the splicing of multiple group II introns in maize mitochondria, and the protein-protein interaction between it and Zm-mCSF1 might allow the formation of large macromolecular splicing complexes.

Multiple giant coronary artery aneurysms combined with right coronary artery-pulmonary artery fistula: a case report.

BACKGROUND: The combination of multiple giant coronary artery aneurysms (CAAs) and right coronary artery (RCA) to pulmonary artery (PA) fistula is extremely rare and the patients with CAAs may suffer from several fatal complications. We herein describe a 60-year-old female with hemodynamic instability who was diagnosed with multiple giant CAAs combined with RCA-PA fistula. CASE PRESENTATION: The patient, a 60-year-old female, presented to the emergency room because of progressive exertional chest distress and fatigue. The transthoracic echocardiography (TTE), coronary computed tomography angiography (CTA) and invasive coronary angiography confirmed the existence of multiple giant CAAs and RCA-PA fistula. Laboratory examinations for systemic vasculitis and infectious diseases demonstrated no abnormalities and work-up for childhood and family history were negative. We have performed a successful surgical treatment for this patient. The patient's restrictive cardiac dysfunction was improved after debriding the advanced thrombi in aneurysm sac and ligating the fistulous vessel between the native RCA and PA. The postoperative pathologic examination of the aneurysmal wall revealed loss of smooth muscle cells in the media with local mucoid degeneration, no chronic inflammation, sclerosis and IgG4 were observed. CONCLUSIONS: The treatment decision-making process should depend upon the patients' specific situations. Our case suggests the surgical intervention should be accepted as the preferred treatment for giant CAAs with restrictive cardiac dysfunction.

[Correlation of Krebs Von Den Lungen-6（KL-6） Expression and Perioperative Pulmonary Function in Adults Receiving Continuing Low-volume Ventilation during Valve Surgery].

OBJECTIVE: To investigate the influence of continuous low-volume ventilation in cardiopulmonary bypass (CPB) on the expression of krebs von den lungen-6 (KL-6) and perioperative pulmonary function in adults undergoing valve surgery. METHODS: From 2017 Sept. to 2018 Jan., 60 patients who received valve replacement surgery were prospectively comprised in this study. We randomly allocated these patients into control group ( n=30, non-ventilation) and experimental group ( n=30, continuous low-volume ventilation during CPB). At different perioperative time points, we identified the dynamic changes of pulmonary function and biomarkers which expressed in serum and bronchoalveolar lavage fluid (BALF). Meanwhile, we also compared perioperative clinical outcomes of the two groups. RESULTS: The expression of serum KL-6 and BALF KL-6 in the two groups were both statistically significant（ P＜0.05）. The oxygenation index increased with time and reached to the top point at T 1, then subsequently decreased with time ( P＜0.05). The alveolar-arterial oxygen tension difference (P A-aO 2) in the two groups fluctuate with time, the differences have no statistical significance. According to linear correlation, the serum KL-6 level was negatively correlated with oxygenation indexes ( r=－0.525, P=0.003), while it was positively correlated with P A-aO 2 ( r=0.489, P=0.006). There were no significant differences between the two groups in clinical outcomes. CONCLUSION: Continuing low-volume ventilation during CPB could decrease the expression level of KL-6, while it has no significant influence on perioperative outcomes.

[Risk Factors for Adverse Aortic Remodeling in Post-TEVAR Patients with Acute Stanford B Aortic Dissection].

OBJECTIVES: To assess morphological changes of distal aorta and the risk factors for adverse aortic remodeling inpost-TEVAR（thoracic endovascular aortic repair） patients with acute Stanford B aortic dissection. METHODS: We retrospectively investigated the patients who underwent TEVAR for a type B dissection between October 2005 and December 2015. CT angiogram (CTA) was obtained for each patients preoperatively, postoperatively and during the post-operational follow-up. Based on Criadol partition principle, we divided the aorta into descending thoracic aorta area, suprarenal abdominal aorta area, infrarenal abdominal aorta area and iliac artery area, and evaluated the distribution of aortic tears and the form of true and false lumen in different aortic partition. Univariate and multivariate logistic regression analyses were used to analyze the risk factors affecting distal aortic remodeling. RESULTS: Of 216 patients (mean follow-up (3.9±2.1) years) who were regularly followed up in our center, 47 patients (21.8%) occurred adverse remodeling in distal aorta. Univariate logistic regression indicated that abnormal aortic wall structure (Marfan's syndrome) and patent false lumen (existence of distal tears, decreased complete false lumen thrombosis) were associated with distal aortic adverse remodeling. Multivariate logistic regression showed that more tears in descending thoracic aorta area ( OR=1.36, 95% CI=1.12-1.58, P=0.005) and less tears in infrarenal abdominal aorta area ( OR=0.49, 95% CI=0.22-0.71, P<0.001) were independent risk factors affecting remodeling in distal aorta after TEVAR. CONCLUSIONS: Aortic wall structure abnormalities, a patent false lumen, more tears in descending thoracic aorta area, less tears in infrarenal abdominal aorta area are independent risk factors for adverse aortic remodeling in post-TEVAR patients with acute Stanford B aortic dissection.

[Effect of cultivation substrate on growth and active component contents of Anoectochilus roburghii from three different origins].

Three different origins of Anoectochilus roburghii were used as experimental materials to study the effect of three different substrate( peat soil-river sand-peanut shell) radio on survival rate,plant height,stem diameter,plant fresh weight,root number,the longest root length,root diameter,and the contents of polysaccharide,flavonoids,and polyphenol. The results showed that when the substrate ratio was 4 ∶2 ∶2,the survival rate of A. roburghii from different origins was the highest,and the plant height,stem diameter,plant fresh weight,the longest root length and root diameter were also the largest. The cultivation substrate had no significant effect on the polysaccharide content of A. roxburghii and A. formosanus. When the substrate ratio was 4 ∶ 2 ∶ 2,the polysaccharide content of A.chapaensis was significantly lower than that of the other two combinations. When the substrate ratio was 4 ∶2 ∶1,the flavonoid content of A. formosanus was higher than that of the other two combinations. When the substrate ratio was 4 ∶2 ∶2,A. formosanus and A. chapaensis had higher polyphenol content.