Characterization and implementation of the MarathonRT template-switching reaction to expand the capabilities of RNA-Seq.

End-to-end RNA sequencing methods that capture 5'-sequence content without cumbersome library manipulations are of great interest, particularly for analysis of long RNAs. While template-switching methods have been developed for RNA sequencing by distributive short-read RTs, such as the MMLV RT enzymes used in SMART-Seq methods, they have not been adapted to leverage the power of ultraprocessive RTs, such as those that derive from group II self-splicing introns. To facilitate this transition, we dissected the individual processes that guide the enzymatic specificity and efficiency of the multi-step template switching reaction carried out by RT enzymes, in this case, by a well-characterized enzyme known as MarathonRT. Remarkably, this is the first study of its kind, for any RT. First, we characterized and optimized the enzymatic nontemplated addition (NTA) reaction that occurs when the RT enzyme extends past the RNA 5'-terminus, and we determined the nucleotide specificity of the NTA reaction. We then evaluated the binding specificity of specialized template-switching oligonucleotides, optimizing their sequences and chemical properties to guide efficient template switching reaction. Having dissected and optimized these individual steps, we then unified them into a procedure for performing RNA sequencing with MarathonRT enzymes, using a well-characterized RNA reference set. The resulting reads span a six-log range in transcript concentration and accurately represent the input RNA identities in both length and composition. We also performed RNA-seq starting from total human RNA and poly(A)-enriched RNA, with short and long-read sequencing demonstrating that MarathonRT enhances the discovery of unseen RNA molecules by conventional RT. Altogether, by employing mechanistic enzymology on RT enzymes and using them to modify RNA-seq technologies, we have generated a new pipeline for rapid, accurate sequencing of complex RNA libraries containing mixtures of long RNA transcripts.

Exploration of cardiopulmonary resuscitation teamwork training for maternal cardiac arrest using the SimMan intelligent simulation platform: A simulation teaching study.

Background and Aims: Maternal cardiac arrest is the most urgent clinical event in obstetrics and can lead to serious consequences, such as maternal or fetal death. Therefore, the training of team cardiopulmonary resuscitation (CPR) skills for obstetricians is essential. The aim of this study was to investigate the effect of applying intelligent simulation to CPR in maternal cardiac arrest teamwork training for obstetricians. Methods: Twenty-four obstetricians who participated in the "Maternal First Aid Workshop," organized by our hospital in 2018, were selected as training participants. The SimMan intelligent comprehensive patient simulator was used to train the CPR team collaboration with first-aid skills. Each team participating in the training was assessed before and after the training using a questionnaire survey. Results: The evaluation of the results after the training showed that all four teams were qualified and that the timing of the cesarean section was 100% correct. The mean score, team collaboration score, and chest compression fraction were significantly higher than before training. Teamwork CPR assessment time, interruption time of chest compressions, and artificial airway establishment time were significantly shorter than before training. The questionnaire survey showed that 95.8% of the physicians reported that the training was rewarding and helpful to their clinical work, and 100% of the physicians believed that obstetricians require similar training. Conclusion: Using the SimMan intelligent comprehensive patient simulator to train obstetricians for CPR of maternal cardiac arrest teamwork first-aid skills can significantly improve the training effect, clinical first-aid skills, and teamwork awareness.

RT-based Sanger sequencing of RNAs containing complex RNA repetitive elements.

Although next-generation sequencing (NGS) technologies have revolutionized our ability to sequence DNA with high-throughput, the chain termination-based Sanger sequencing method remains a widely used approach for DNA sequence analysis due to its simplicity, low cost and high accuracy. In particular, high accuracy makes Sanger sequencing the "gold standard" for sequence validation in basic research and clinical applications. During the early days of Sanger sequencing development, reverse transcriptase (RT)-based RNA sequencing was also explored and showed great promise, but the approach did not acquire popularity over time due to the limited processivity and low template unwinding capability of Avian Myeloblastosis Virus (AMV) RT, and other RT enzymes available at the time. RNA molecules have complex features, often containing repetitive sequences and stable secondary or tertiary structures. While these features are required for RNA biological function, they represent strong obstacles for retroviral RTs. Repetitive sequences and stable structures cause reverse transcription errors and premature primer extension stops, making chain termination-based methods unfeasible. MarathonRT is an ultra-processive RT encoded group II intron that can copy RNA molecules of any sequence and structure in a single cycle, making it an ideal RT enzyme for Sanger RNA sequencing. In this chapter, we upgrade the Sanger RNA sequencing method by replacing AMV RT with MarathonRT, providing a simple, robust method for direct RNA sequence analysis. The guidance for troubleshooting and further optimization are also provided.

End-to-end RT-PCR of long RNA and highly structured RNA.

RNA molecules play important roles in numerous normal cellular processes and disease states, from protein coding to gene regulation. RT-PCR, applying the power of polymerase chain reaction (PCR) to RNA by coupling reverse transcription with PCR, is one of the most important techniques to characterize RNA transcripts and monitor gene expression. The ability to analyze full-length RNA transcripts and detect their expression is critical to decipher their biological functions. However, due to the low processivity of retroviral reverse transcriptases (RTs), we can only monitor a small fraction of long RNA transcripts, especially those containing stable secondary and tertiary structures. The full-length sequences can only be deduced by computational analysis, which is often misleading. Group II intron-encoded RTs are a new type of RT enzymes. They have evolved specialized structural elements that unwind template structures and maintain close contact with the RNA template. Therefore, group II intron-encoded RTs are more processive than the retroviral RTs. The discovery, optimization and deployment of processive group II intron RTs provide us the opportunity to analyze RNA transcripts with single molecule resolution. MarathonRT, the most processive group II intron RT, has been extensively optimized for processive reverse transcription. In this chapter, we use MarathonRT to deliver a general protocol for long amplicon generation by RT-PCR, and also provide guidance for troubleshooting and further optimization.

Effects of Cadmium Stress on Bacterial and Fungal Communities in the Whitefly Bemisia tabaci.

Heavy metal contamination is among the most prominent environmental problems in China, posing serious threats to both ecosystem and human health. Among the diverse heavy metal contaminants, cadmium is the most serious. The whitefly Bemisia tabaci is a cosmopolitan pest capable of causing severe damage to a broad range of agricultural crops, especially vegetables. At present, little is known about the effects of cadmium stress on B. tabaci, including on its bacterial and fungal communities. In the current study, we investigated the effects of cadmium on bacterial and fungal communities in whiteflies. Meta-barcode sequencing of the 16S rRNA gene revealed that the whitefly bacterial community contained 264 operational taxonomic units (OTUs) belonging to 201 known genera and 245 known species. The top five most frequent bacterial genera were Rickettsia, Rhodococcus, Candidatus Portiera, Candidatus Hamiltonella, and Achromobacter. Meta-barcode sequencing of the fungal ITS locus revealed that the whitefly fungal community contained 357 OTUs belonging to 187 known genera and 248 known species. The top five most frequent fungal genera were Wallemia, unclassified_f_Dipodascaceae, Apiotrichum, Penicillium, and unclassified_o_Saccharomycetales. Cadmium exposure reduced the fungal OTU richness but increased the bacterial Shannon and Simpson diversity indices in whiteflies. In addition, upon exposure to cadmium, the microbial community composition in whiteflies changed significantly, with increased prevalence of the bacterial genera Rhodococcus and Exiguobacterium and fungal genus Wallemia. Our results indicate that the whitefly microbiota likely contributed to their adaptation and resistance to cadmium and suggested that whiteflies may contain microbes that could help remediate cadmium contamination in natural environments and agricultural fields.

Understanding Acute Respiratory Distress Syndrome in High-Altitude Environments: A Comprehensive Review of Diagnosis and Treatment.

Approximately 2% of the global population lives above 1500 m, where low atmospheric pressure, decreased oxygen levels, harsh cold and dry conditions, strong radiation, and the effects of climate change present significant health challenges. Residents of these high-altitude areas display physiological adaptions, including smaller body size, enlarged ribs, improved oxygen delivery in hypoxic conditions, and adjustments in oxygen utilization and metabolism. Both acute and chronic hypoxia prevalent in such regions can trigger various diseases by stimulating hypoxia-inducible factors, boosting inflammatory responses, and impairing mitochondrial function.Acute Respiratory Distress Syndrome (ARDS) - a critical respiratory condition associated with high morbidity and mortality - occurs more frequently among the health risks in these environments. Hypoxia is a critical predisposing and aggravating factor for high-altitude ARDS. Despite similarities with its low-altitude counterpart, ARDS in high-altitude areas displays unique pathophysiology and clinical manifestations due to the specific environmental conditions.This review aims to shed light on how high-altitude environments influence the diagnosis and treatment of ARDS, providing a comprehensive understanding of the distinct challenges inherent to these regions.

Management of massive airway hemorrhage associated with extracorporeal membrane oxygenation: A retrospective case series study.

Background and Aims: Extracorporeal membrane oxygenation (ECMO) is an important means of treating patients with respiratory failure. Massive airway hemorrhage is a rare complication of ECMO, with high mortality. The aim of this study was to provide a reference for improving the success rate of treatment of this complication by analyzing and summarizing patient clinical data. Methods: We searched PubMed, Medline, and EMBASE databases for case reports of massive airway bleeding associated with ECMO from January 2000 to January 2022 and included one case treated at our facility. All patients were disconnected from the ventilator, and the endotracheal tube was clamped during treatment, resulting in complete airway packing for hemostasis. The clinical data of these patients were analyzed. Results: Through searching and further screening, two works of literature reported four cases that met our inclusion criteria. Including our patient's case, five patients were included in this study (four adults and one neonate). The longest ECMO treatment time before bleeding was 14 days, and the shortest was 20 min. In all patients, conservative treatment was ineffective after a major airway hemorrhage. They were disconnected from the ventilator and the tracheal tube was clamped for 13-72 h. The four adult patients underwent bronchial artery embolization in the interventional radiology suite. All patients' bleeding stopped after treatment; they were successfully weaned off ECMO and discharged. Conclusions: Treatment measures to disconnect the ventilator and clamp the endotracheal tube with full support from ECMO are feasible for massive airway bleeding associated with ECMO. Early bronchial arteriography and embolization can prevent rebleeding.

Factors affecting continuous renal replacement therapy duration in critically ill patients: A retrospective study.

INTRODUCTION: This study aimed to analyze the factors affecting continuous renal replacement therapy (CRRT) duration in critically ill patients and provide a reference for clinical treatment. MATERIAL AND METHODS: We divided patients into regional citrate anti-coagulation (RCA) and low-molecular-weight-heparin (LMWH) groups according to the anti-coagulation method and collected the relevant data, to analyze the factors associated with CRRT time. RESULTS: Compared with the LMWH group, the RCA group had a longer mean treatment time (55.36 ± 22.57 vs. 37.65 ± 27.09 h, p < 0.001), lower transmembrane pressure, and lower filter pressure, regardless of vascular access site. Multivariable linear regression analysis showed a significant correlation between anti-coagulation patterns, filter pressure at CRRT discontinuation, nurses' level of intensive care unit experience, pre-machine fibrinogen level, and CRRT time. CONCLUSION: Anti-coagulation is the most important factor affecting CRRT duration. Filter pressure, nurses' level of intensive care unit experience, and fibrinogen level also affecting CRRT duration.

Massive Airway Hemorrhage in Severe COVID-19 and the Role of Endotracheal Tube Clamping.

Background: Venovenous extracorporeal membrane oxygenation (VV-ECMO) has been widely used in treating patients with coronavirus disease 2019 (COVID-19) with severe respiratory failure. However, there are few reports of the successful treatment of patients with massive airway hemorrhage in severe COVID-19 during VV-ECMO treatment. Methods: We analyzed the treatment process of a patient with a massive airway hemorrhage in severe COVID-19, who underwent prolonged VV-ECMO treatment. Results: A 59-year-old female patient was admitted to the intensive care unit after being confirmed to have severe acute respiratory syndrome coronavirus 2 infection with severe acute respiratory distress syndrome. VV-ECMO, mechanical ventilation, and prone ventilation were administered. Major airway hemorrhage occurred on day 14 of ECMO treatment; conventional management was ineffective. We provided complete VV-ECMO support, discontinued anticoagulation, disconnected the ventilator, clipped the tracheal intubation, and intervened to embolize the descending bronchial arteries. After the airway hemorrhage stopped, we administered cryotherapy under bronchoscopy, low-dose urokinase locally, and bronchoalveolar lavage in the airway to clear the blood clots. The patient's condition gradually improved; she underwent ECMO weaning and decannulation after 88 days of VV-ECMO treatment, and the membrane oxygenator was changed out four times. She was successfully discharged after 182 days in hospital. Conclusion: Massive airway hemorrhage in patients with severe COVID-19 and treated with ECMO is catastrophic. It is feasible to clamp the tracheal tube with the full support of ECMO. Notably, bronchoscopy with cryotherapy is effective for removing blood clots.

Analysis of the extracorporeal anticoagulation effect of modified citrate infusion during continuous renal replacement therapy in critically ill patients.

INTRODUCTION: To analyze the anticoagulation effect of different local infusion methods of citrate underwent continuous renal replacement therapy (CRRT) in critically ill patients. METHODS: The study adopted a single-centre retrospective design. Critically ill patients were divided into conventional group and modified group based on the infusion methods of citrate. RESULTS: The modified group had a longer mean treatment time (67.67 ± 18.69 hours vs. 52.11 ± 24.26 hours, p = 0.007), a lower transmembrane pressure (147.77 ± 66.85 cm H2 O vs. 200.63 ± 118.66 cm H2 O, p = 0.038), fewer citrate bag replacements (1.43 ± 0.50 times vs. 10.60 ± 3.19 times, p < 0.001), and more steady ionized calcium at the venous end (0.35 ± 0.06 mmol/L vs. 0.40 ± 0.05 mmol/L, p = 0.006) compared to the conventional group patients, with statistically significant differences. The incidences of citrate accumulation and tubing coagulation were marginally lower in the modified group. CONCLUSION: The modified local citrate infusion method can prolong treatment time, while reducing both the nursing workload and the occurrence of citrate accumulation.

[Dead space fraction for treatment guidance and prognosis evaluation of acute respiratory distress syndrome].

Acute respiratory distress syndrome (ARDS) is a common cause of critical illness and high mortality from respiratory failure. Increased dead space fraction (VD/VT) was independently associated with lung injury and mortality of ARDS. VD/VT is readily obtained by bedside measurements of arterial blood gas and end-tidal carbon dioxide. Early attention and application of VD/VT as an indicator will help to better understand the pathophysiological of ARDS, guide clinical treatment, and better assess the severity and clinical prognosis of the disease.

Hemorrhagic Fever with Renal Syndrome Complicated with Rhino Mucormycosis: A Case Report.

Mucormycosis (MCR) is a rare but aggressive fungal disease. Rhino-orbito-cerebral mucormycosis is the most common clinical form of MCR infection, and sinonasal inoculation is the primary site of infection. The morbidity and mortality rates associated with MCR remain high. In this case report, we describe the successful use of amphotericin B in a 40-year-old male with hemorrhagic fever with renal syndrome (HFRS) complicated by rhinomucormycosis. This case report provides evidence for the successful treatment of HFRS.

Bacterial and fungal communities within and among geographic samples of the hemp pest Psylliodes attenuata from China.

The hemp flea beetle Psylliodes attenuata (Coleoptera: Chrysomelidae: Psylliodes) is a common pest of Cannabis sativa, including cultivars of both medicinal marijuana and industrial hemp. Both the larval and adult stages of this beetle can cause significant damages to C. sativa, resulting in substantial crop losses. At present, little is known about the bacterial and fungal community diversity among populations of this pest insect. In the present study, we obtained P. attenuata samples from nine field sites representing broad industrial hemp productions in China and analyzed their microbial communities using DNA metabarcoding. Bacterial sequences of all the samples were assigned to 3728 OTUs, which belonged to 45 phyla, 1058 genera and 1960 known species. The most common genera were Rickettsia, Wolbachia, and Candidatus_Brownia. Fungal sequences of all the samples were assigned to 910 OTUs, which belonged to 9 phyla, 308 genera and 464 known species. The most common fungal genera were Cladosporium, Cutaneotrichosporon, and Aspergillus. Principal coordinate analysis revealed a significant difference in the bacterial and fungal community structure among the nine P. attenuata populations. Understanding the microbial symbionts may provide clues to help develop potential biocontrol techniques against this pest.

Ancestral archaea expanded the genetic code with pyrrolysine.

The pyrrolysyl-tRNA synthetase (PylRS) facilitates the cotranslational installation of the 22nd amino acid pyrrolysine. Owing to its tolerance for diverse amino acid substrates, and its orthogonality in multiple organisms, PylRS has emerged as a major route to install noncanonical amino acids into proteins in living cells. Recently, a novel class of PylRS enzymes was identified in a subset of methanogenic archaea. Enzymes within this class (ΔPylSn) lack the N-terminal tRNA-binding domain that is widely conserved amongst PylRS enzymes, yet remain active and orthogonal in bacteria and eukaryotes. In this study, we use biochemical and in vivo UAG-readthrough assays to characterize the aminoacylation efficiency and substrate spectrum of a ΔPylSn class PylRS from the archaeon Candidatus Methanomethylophilus alvus. We show that, compared with the full-length enzyme from Methanosarcina mazei, the Ca. M. alvus PylRS displays reduced aminoacylation efficiency but an expanded amino acid substrate spectrum. To gain insight into the evolution of ΔPylSn enzymes, we performed molecular phylogeny using 156 PylRS and 105 pyrrolysine tRNA (tRNAPyl) sequences from diverse archaea and bacteria. This analysis suggests that the PylRS•tRNAPyl pair diverged before the evolution of the three domains of life, placing an early limit on the evolution of the Pyl-decoding trait. Furthermore, our results document the coevolutionary history of PylRS and tRNAPyl and reveal the emergence of tRNAPyl sequences with unique A73 and U73 discriminator bases. The orthogonality of these tRNAPyl species with the more common G73-containing tRNAPyl will enable future efforts to engineer PylRS systems for further genetic code expansion.

Direct tracking of reverse-transcriptase speed and template sensitivity: implications for sequencing and analysis of long RNA molecules.

Although reverse-transcriptase (RT) enzymes are critical reagents for research and biotechnology, their mechanical properties are not well understood. In particular, we know little about their relative speed and response to structural obstacles in the template. Commercial retroviral RTs stop at many positions along mixed sequence templates, resulting in truncated cDNA products that complicate downstream analysis. By contrast, group II intron-encoded RTs appear to copy long RNAs with high processivity and minimal stops. However, their speed, consistency and pausing behavior have not been explored. Here, we analyze RT velocity as the enzyme moves through heterogeneous sequences and structures that are embedded within a long noncoding RNA transcript. We observe that heterogeneities in the template are highly disruptive to primer extension by retroviral RTs. However, sequence composition and template structure have negligible effects on behavior of group II intron RTs, such as MarathonRT (MRT). Indeed, MRT copies long RNAs in a single pass, and displays synchronized primer extension at a constant speed of 25 nt/sec. In addition, it passes through stable RNA structural motifs without perturbation of velocity. Taken together, the results demonstrate that consistent, robust translocative behavior is a hallmark of group II intron-encoded RTs, some of which operate at high velocity.

Genetic Diversity and Population Structure of Fusarium commune Causing Strawberry Root Rot in Southcentral China.

Strawberry plants and fruits are vulnerable to infections by a broad range of pathogens and pests. However, knowledge about the epidemiology of pathogens causing strawberry diseases is limited. In this study, we analyzed Fusarium commune, a major fungal pathogen causing strawberry root rot, from diseased strawberry root tissues in southcentral China. A total of 354 isolates were obtained from 11 locations that spanned about 700 km from both south to north and east to west. Multilocus genotypes of all isolates were obtained using seven polymorphic simple sequence repeat markers developed in this study. Our analyses revealed significant genetic diversity within each of the 11 local populations of F. commune. STRUCTURE analysis revealed that the optimal number of genetic populations for the 354 strains was two, with most local geographic populations containing isolates in both genetic clusters. Interestingly, many isolates showed allelic ancestry to both genetic clusters, consistent with recent hybridization between the two genetic clusters. In addition, though alleles and genotypes were frequently shared among local populations, statistically significant genetic differentiations were found among the local populations. However, the observed F. commune population genetic distances were not correlated with geographic distances. Together, our analyses suggest that populations of F. commune causing strawberry root rot are likely endemic to southcentral China, with each local population containing shared and unique genetic elements. Though the observed gene flow among geographic regions was relatively low, human activities will likely accelerate pathogen dispersals, resulting in the generation of new genotypes through mating and recombination.

Prognosis of Patients With Brainstem Glioblastoma Based on "age, surgery and radiotherapy": A SEER Database Analysis.

Introduction: Primary brainstem glioma is a rare tumor with a dismal prognosis that poses significant treatment challenges. The purpose of the current study is to identify and determine prognostic factors associated with survival in high-grade brainstem glioma patients. Methods: We gathered the data from the SEER database for the duration of years from 1973 to 2016 to examine the survival of patients particularly reported with the high-grade brainstem glioma and subsequently ascertained the potential impact of demographic features, tumor, and clinical characteristics on the overall survival of these patients. The survival patterns were assessed using Kaplan-Meier curves and Cox proportional hazards models. Propensity score matching (PSM) analysis was performed between patients with or without radiation therapy based on age and surgical resection to investigate the effect of radiotherapy on overall survival (OS). Results: A total 232 patient's data were obtained from the SEER database and included in this study. The median overall survival was 8 months. Kaplan-Meier survival analysis delineated that the patients who were in younger age (P = .001) and underwent surgery (P = .001) exhibited typically a better prognosis. Among 232 patients, a total of 204 patients were categorized as radiotherapy group (RG) received radiation therapy whereas 28 patients were considered as nonradiotherapy group (NRG), who were not receiving radiotherapy. Radiotherapy was associated with an improvement in the overall survival without statistical significance (P = .104). PSM was performed between RG and NRG based on age and surgical resection. After the PSM, 56 patients were included. Overall Survival was significantly different between both groups (P = .038). Conclusion: Furthermore, the patients with high-grade brain glioma who received "both radiotherapy and chemotherapy" exhibited significantly longer survival compared to the patients who received chemotherapy alone. Multivariate analysis showed that treatment with surgery and radiotherapy were considered as the independent prognostic factors (P < .05).

Disseminated Hematogenous Tuberculosis Following in vitro Fertilization-Embryo Transfer: A Case Report.

This study aimed to analyze the diagnosis and treatment process of patients with hematogenous disseminated pulmonary tuberculosis after treatment with in vitro fertilization-embryo transfer (IVF-ET). We retrospectively analyzed the clinical data, including imaging and etiological data, the use of antimicrobials, metagenomic next-generation sequencing (mNGS) results, and the treatment process, of a patient who underwent IVF-ET due to an obstruction in the fallopian tube; after the treatment, she developed a persistent fever with shortness of breath and suffered a spontaneous abortion. Due to the failure of other treatment modalities, fiber optic bronchoscopy was performed, and the alveolar lavage fluid was obtained for mNGS. Tests for Mycobacterium tuberculosis and rifampicin resistance (Xpert MTB/RIF) showed positive and negative results, respectively. Subsequently, anti-tuberculosis treatment with isoniazid, rifampicin, pyrazinamide, and ethambutol was administered. After the patient's condition improved, she was transferred to a specialized tuberculosis hospital for further treatment, where she died one month later from multiple organ failure. From this case, we conclude that clinicians should remain highly vigilant for pulmonary infection with M. tuberculosis in pregnant women, particularly in patients treated with IVF-ET, and check for its presence as soon as possible.