Exploring Stigma Experience and Coping Strategies Among Women Living with HIV/AIDS in China: A Phenomenological Study.

Purpose: As of the end of 2022, over 20 million women worldwide, aged 15 and above, are living with HIV. Stigma remains a formidable barrier for women living with HIV/AIDS, hindering their access to healthcare and exacerbating health disparities. Indeed, some women living with HIV/AIDS can successfully confront and overcome stigma. There remains a paucity of qualitative research exploring the stigma coping strategies of women living with HIV/AIDS in China. This study was aimed to gain the deeper understanding of stigma experienced by women living with HIV/AIDS and coping strategies. Patients and Methods: We recruited diverse participants using snowball sampling and purposive sampling. Semi-structured personal in-depth interviews were conducted with 30 women living with HIV/AIDS from December 2022 to June 2023. The samples were from four HIV/AIDS designated hospitals. The data were analyzed using the Colaizzi seven-step model. Results: The experiences of stigma among women living with HIV/AIDS included family role (wife/mother/grandmother) collapse and disgusted by family, resignation in being shunned by others, helplessness due to social exclusion, grief at being devaluated, and resentment for experiencing injustice. The coping strategies used to deal with stigma included concealing their conditions, avoiding socialization, and attempting to retaliate against society. Conclusion: Healthcare professionals are recommended to offer women living with HIV/AIDS effective emotional support and guidance to cope with stigma. The study highlights the stigma they face, providing valuable evidence for policymakers. Recommendations emphasize the importance of developing services addressing both physical and psychological needs of women living with HIV/AIDS.

Reliability, validity, and responsiveness of the simplified Chinese version of the knee injury and Osteoarthritis Outcome Score in patients after total knee arthroplasty.

Objectives: The Knee Injury and Osteoarthritis Outcome Score (KOOS) has been utilized to evaluate short- and long-term outcomes in individuals following knee injuries, such as those with anterior cruciate ligament reconstruction and knee osteoarthritis, but has not yet been applied to individuals undergoing total knee arthroplasty (TKA) in China. The aim of this study was to assess the psychometric properties of the Simplified Chinese version of the KOOS in Chinese individuals undergoing TKA. Methods: This study distributed 170 questionnaires, and assessed the KOOS of the participants, along with requiring them to complete the Short Form 36 (SF-36) survey. There were 35 participants completed a test-retest reliability survey with a 24-h interval, 129 participants completed a pre - surgery survey, and 119 individuals completed a post - surgery survey 6 weeks after the surgery. The following tests were conducted: Cronbach's alpha (α) to assess internal consistency, intraclass correlation coefficient (ICC) to evaluate test-retest reliability, Spearman's correlation coefficient (ρ) to examine construct validity, effect size (ES) to detect measure responsiveness, minimal detectable change (MDC) to assess measurement errors. Floor and ceiling effects (<15%) were also asses evaluated. Results: The simplified Chinese version of the KOOS showed good test-retest reliability in participants after TKA, with an ICC of 0.82-0.97 (95% CI). The internal consistency of the five subscales of the KOOS was good (Cronbach's α = 0.70-0.96). No floor or ceiling effects were found. Regarding construct validity, a strong positive correlation was found between each of the three KOOS subscales (activities of daily living, knee-related Quality of Life, and sport and recreation subscales) and the general health and bodily pain subscales of the SF-36 (0.53 < ρ < 0.61). The subscales of the simplified Chinese version of the KOOS showed responsiveness (ES: 0.68 to 0.86) before and after 6 weeks of physical treatment. The MDC ranged from 10.28 to 23.24. Conclusions: The Chinese version of the KOOS showed good psychometric properties and was found to be valid, reliable, and simple as an assessment tool for symptoms, pain, activity of daily living, sports and recreational activity and quality of life for the Chinese population suffering from TKA.

Gender differences in symptom burden among people living with HIV/AIDS receiving antiretroviral therapy in Yunnan, China.

ABSTRACTLittle is known about gender differences in the symptom burden of people living with HIV/AIDS (PLWHA) on antiretroviral therapy in China. This study was conducted based on a biopsychosocial-medical model to describe gender differences in symptom burden among 1035 PLWHA in Yunnan Province, China. After propensity score matching, 798 PLWHA were included in this analysis. Feeling stressed, poor sleep, and memory loss were the most burdensome symptoms among men, while feeling stressed, memory loss, and dizziness were the most burdensome symptoms among women. Among men PLWHA, factors associated with symptom burden were being of the ethnic minority, CD4 count ≥ 500 cells/mm3, physical functioning, and social support. Among women PLWHA, factors associated with symptom burden were being an inpatient, physical functioning, psychological functioning, and social support. Our findings suggest that healthcare providers need to take into account gender differences when developing optimal prevention, treatment, and care programs that provide individualized care to reduce patients' symptom burden.

The simplified Chinese version of the Knee Injury and Osteoarthritis Outcomes Score (KOOS) in individuals with knee osteoarthritis for mainland China: the study of reliability and validity.

BACKGROUND: The Knee Injury and Osteoarthritis Outcomes Score (KOOS) is a free clinical tool commonly used to evaluate the symptoms and functional status of patients with knee injury. For people who speak Chinese, the Hong Kong Chinese and Singapore Chinese versions are preferred. However, variations in the Chinese language and culture are influenced by the country's geography. KOOS for Mainland China has not been reported. Therefore, the current study was to cross-culturally translate the original English version into a simplified Chinese version and to investigate its psychometric properties. METHODS: The simplified Chinese KOOS was obtained through forward-backward translation according to appropriate guidelines. A total of 158 individuals with knee osteoarthritis (KOA) were recruited from 13 hospitals in China to examine the psychometric properties. The test-retest questionnaire was performed at an interval of 5-7 days. Test-retest reliability and internal consistency were evaluated using the intraclass correlation coefficient (ICC) and Cronbach's alpha, respectively. The data of the first test were used to analyse the construct validity of the simplified Chinese KOOS and Chinese SF-36 through convergent and discriminant validity using Spearman's correlation coefficient. RESULTS: Cross-cultural translation exhibited minor cultural differences, and the questionnaire was well understood by the patients. The data from 128 patients, used for the test-retest reliability study, showed good to excellent reliability, with an ICC of 0.808-0.976 for all KOOS subscales. The Cronbach's alpha for all subscales ranged from 0.757 to 0.970, indicating acceptable internal consistency. There was a low-to-high correlation between the five domains of the simplified Chinese version of the KOOS and all domains of the SF-36 in construct validity. CONCLUSION: The simplified Chinese KOOS demonstrated acceptable reliability and validity. In clinical practice and research, this version can help provide valuable information on health-related quality of life for Chinese individuals with KOA in mainland China.

Combating Stigma and Health Inequality of Monkeypox: Experience from HIV.

With Western Pacific Region reporting more imported cases, the World Health Organization (WHO) declared monkeypox a public health emergency of international concern in July. Currently, we are now at the beginning of forming stigmatization toward monkeypox. Based on the experience of combating HIV stigmatization for over 30 years, in this comment, we proposed measures based on the three stages of the stigma formation process. We believe the paper may be of particular interest to the readers of your journal as it offered implications for both healthcare professionals and researchers in Western Pacific Region.

Identifying subtypes of HIV/AIDS-related symptoms in China using latent profile analysis and symptom networks.

The identification of subgroups of people living with HIV in China based on the severity of symptom clusters and individual symptoms is crucial to determine group-specific symptom management strategies. Participants reported 27 highly prevalent HIV/AIDS-related symptoms. Latent profile analysis based on symptom severity was used to identify person-centered subtypes of HIV/AIDS-related symptoms. Symptom networks were compared among subgroups identified by latent profile analysis. A total of 2927 eligible people living with HIV (PWH) were included in the analysis. Five profiles were identified: "Profile 1: all low symptom severity" (n2 = 2094, 71.54%), "Profile 2: medium symptom severity with syndemic conditions" (n3 = 109, 3.72%), "Profile 3: medium symptom severity with low functional status" (n1 = 165, 5.64%), "Profile 4: medium symptom severity in transitional period" (n4 = 448, 15.31%), and "Profile 5: all high symptom severity" (n5 = 111, 3.79%). Except for Profile 1 and Profile 5, the symptom severity was similar among the other three profiles. Profiles 1 (2.09 ± 0.52) and 4 (2.44 ± 0.66) had the smallest ∑s values, and Profiles 2 (4.38 ± 1.40) and 5 (4.39 ± 1.22) had the largest ∑s values. Our study demonstrates the need for health care professionals to provide PWH with group-specific symptom management interventions based on five profiles to improve their physical and psychological well-being. Future studies should be conducted in different contexts using different symptom checklists to further validate our results.

Unraveling the central and bridge psychological symptoms of people living with HIV: A network analysis.

Background: People living with HIV (PLWH) experience multiple psychological symptoms. Few studies have provided information on central and bridge psychological symptoms among PLWH. This information has implications for improving the efficiency and efficacy of psychological interventions. Our study aimed to identify the central and bridge psychological symptoms of PLWH and to explore the interconnectedness among symptoms and clusters. Methods: Our study used data from the HIV-related Symptoms Monitoring Survey, a multisite, cross-sectional study conducted during 2017-2021. We used R to visualize the network of 16 symptoms and analyzed the centrality and predictability indices of the network. We further analyzed the bridge symptoms among the three symptom clusters. Results: A total of 3,985 participants were included in the analysis. The results suggested that sadness had the highest strength (r S = 9.69) and predictability (70.7%) compared to other symptoms. Based on the values of bridge strength, feeling unsafe (r bs = 0.94), uncontrollable worry (r bs = 0.82), and self-abasement (r bs = 0.81) were identified as bridge symptoms. We also found a strong correlation between sadness and self-abasement (r = 0.753) and self-loathing and self-blame (r = 0.744). Conclusion: We found that sadness was the central psychological symptom of PLWH, indicating that sadness was the center of the psychological symptom network from a mechanistic perspective and could be a target for intervention. Deactivating bridge symptoms, including "feeling unsafe," "self-abasement," and "uncontrollable worry," could be more effective in preventing symptom activation from spreading (e.g., one symptom activating another).

How Patients with an Uncertain Diagnosis Experience Intolerance of Uncertainty: A Grounded Theory Study.

PURPOSE: To develop a conceptual framework to explain how patients with uncertain diagnoses experience intolerance of uncertainty (IU) in order to achieve an in-depth understanding of the process of facing uncertainty in this population. PATIENTS AND METHODS: A grounded theory study using semi-structured interviews was conducted with 21 patients with uncertain diagnoses in China from December 2018 to March 2019. The coding process followed the procedures of Strauss and Corbin: open coding, axial coding, and selective coding. RESULTS: We developed a conceptual framework called 'Facing an Uncertain Diagnosis'. This framework considers not only the external and internal context of IU generation but also the ongoing process of how IU impacts patients' psychological status, cognitive response, behavior, and decision making. CONCLUSION: We suggest that healthcare professionals should be aware of the high level of IU and its psychological, cognitive, and behavioral manifestations in patients with uncertain diagnoses. Healthcare professionals should also be more cautious in shared decision making with patients with uncertain diagnoses.

Evolving symptom networks in relation to HIV-positive duration among people living with HIV: A network analysis.

OBJECTIVES: To explore and visualize the relationships among multiple symptoms in people living with HIV (PLWH) and compare centrality indices and the density of symptom networks among groups of individuals with different HIV-positive durations. METHODS: We conducted a secondary analysis of data from the HIV-related Symptoms Monitoring Survey conducted in China. Networks were constructed among 27 symptoms. Centrality properties, including strength and closeness, and network density were used to describe relationships among symptoms in 5 different HIV-positive duration groups. RESULTS: The findings showed that PLWH with longer HIV-positive durations did not have more severe symptoms; instead, their symptom networks were denser than those of their newly HIV-diagnosed counterparts (F = 27.073, P < 0.001). Fatigue was the most severe and central symptom in PLWH with an HIV-positive duration <10 years (rS = 7.79-10.09, rB = 18-44, rC = 0.01). Confusion was the most central symptom across the 3 centrality indices (rS = 11.81, rB = 14.00, rC = 0.02) in PLWH who had HIV-positive durations >10 years. CONCLUSION: This study demonstrates a need to include an assessment of PLWH symptom networks as an essential component of HIV care. We recommended evaluating cognitive function and cognitive training as essential components of HIV care for long-term survivors, even in younger populations (aged ≤50 years).

The barriers to evidence-based nursing implementation in mainland China: A qualitative content analysis.

It has been widely recognized that healthcare practices should be based on up-to-date high-quality evidence; however, the implementation of evidence has been a slow process in nursing practice. It is crucial for clinical practitioners to be aware of the barriers to implementing evidence-based nursing. The aim of this study was to describe participants' experiences and thoughts on barriers to implementing evidence-based nursing in mainland China. Forty-five participants came from 45 evidence-based nursing implementation projects carried out in 16 hospitals in mainland China. Data were collected through observation and semi-structured interviews, after which qualitative content analysis was undertaken. Then, the five themes and subthemes were extracted from the data. The five themes were evidence-based, nurse-related, patient-related, setting barriers, and lack of support. In this study, a variety of barriers influencing evidence implementation in the Chinese nursing context were identified and further explored from the perspective of clarifying misunderstandings about evidence-based nursing, the profound influence of Chinese culture on patients' preferences and attitudes, and the lack of professional knowledge of nurses.

Role of security guards in Code Black events in medical and surgical settings: A retrospective chart audit.

The prevalence of security guards in health care settings is growing worldwide. There is a need to explore and understand their role and actions to inform policy and training and support least restrictive practices in health care. The aim of this study was to conduct a retrospective chart audit of security guard logs to investigate security guard involvement in Code Blacks, called in emergency situations of personal threats including patient and/or visitor violence, in medical and surgical wards in a large metropolitan health network in South Australia. Security guards attended 1664 Code Blacks (0.63% of admissions) over the 2.5-year study period. Events were more frequently reported in medical than surgical wards. The most common reasons for security guard attendance were patients threatening/harming staff and patients threatening/harming themselves. The most frequent security guard actions were "Attend only/standby," "Physical restraint," and "Patient located and returned to the ward." The most frequent outcomes were physical restraint, chemical restraint, and de-escalation respectively. Results highlight the imperative that health services maintain and increase efforts to support least restrictive practice through policy directives and staff training.

Understanding Chinese Nursing Education and Practice for Developing International Nursing Partnerships.

Introduction: Modern nursing was introduced into China by Western missionaries in the 19th century; since then, significant changes continued to occur, which provides beneficial areas of international collaboration based on trends in globalization. Methods and Materials: The description was developed through reviews of published literature, policy documents that inform Chinese nursing practice, education, and the firsthand working experiences between American and Chinese nurses and faculty. Results: 82 articles and 13 governmental documents were included. Chinese nursing has undergone significant changes in the organization, quality assessment, and roles requirements in education and practice. International collaboration areas include addressing the severe faculty shortage, maternal child care, elderly care, quality assessment, and educational programs evaluation. Discussion: Informative knowledge of changing Chinese nursing education and practices in the new millennium, the potential areas, and guides for international nursing collaboration would be meaningful to internationally involved faculty and nurses in China and America.

Quantifying Methane and Methanol Metabolism of "Methylotuvimicrobium buryatense" 5GB1C under Substrate Limitation.

Methanotrophic bacteria are a group of prokaryotes capable of using methane as their sole carbon and energy source. Although efforts have been made to simulate and elucidate their metabolism via computational approaches or 13C tracer analysis, major gaps still exist in our understanding of methanotrophic metabolism at the systems level. Particularly, direct measurements of system-wide fluxes are required to understand metabolic network function. Here, we quantified the central metabolic fluxes of a type I methanotroph, "Methylotuvimicrobium buryatense" 5GB1C, formerly Methylomicrobium buryatense 5GB1C, via 13C isotopically nonstationary metabolic flux analysis (INST-MFA). We performed labeling experiments on chemostat cultures by switching substrates from 12C to 13C input. Following the switch, we measured dynamic changes of labeling patterns and intracellular pool sizes of several intermediates, which were later used for data fitting and flux calculations. Through computational optimizations, we quantified methane and methanol metabolism at two growth rates (0.1 h-1 and 0.05 h-1). The resulting flux maps reveal a core consensus central metabolic flux phenotype across different growth conditions: a strong ribulose monophosphate cycle, a preference for the Embden-Meyerhof-Parnas pathway as the primary glycolytic pathway, and a tricarboxylic acid cycle showing small yet significant fluxes. This central metabolic consistency is further supported by a good linear correlation between fluxes at the two growth rates. Specific differences between methane and methanol growth observed previously are maintained under substrate limitation, albeit with smaller changes. The substrate oxidation and glycolysis pathways together contribute over 80% of total energy production, while other pathways play less important roles.IMPORTANCE Methanotrophic metabolism has been under investigation for decades using biochemical and genetic approaches. Recently, a further step has been taken toward understanding methanotrophic metabolism in a quantitative manner by means of flux balance analysis (FBA), a mathematical approach that predicts fluxes constrained by mass balance and a few experimental measurements. However, no study has previously been undertaken to experimentally quantitate the complete methanotrophic central metabolism. The significance of this study is to fill such a gap by performing 13C INST-MFA on a fast-growing methanotroph. Our quantitative insights into the methanotrophic carbon and energy metabolism will pave the way for future FBA studies and set the stage for rational design of methanotrophic strains for industrial applications. Further, the experimental strategies can be applied to other methane or methanol utilizers, and the results will offer a unique and quantitative perspective of diverse methylotrophic metabolism.

Targeted transcriptional profiling of the tumor microenvironment reveals lymphocyte exclusion and vascular dysfunction in metastatic osteosarcoma.

Osteosarcoma (OS) is the most common bone tumor in pediatric and adolescent/young adult patients yet little is known about the microenvironment that supports this aggressive disease. We have used targeted gene expression profiling and immunohistochemistry to characterize the microenvironment of metastatic and non-metastatic OS specimens from pediatric patients exhibiting poor histologic response to chemotherapy. Our results indicate that metastatic specimens exhibit lymphocyte exclusion as T cells are confined to the periphery of the pulmonary lesions. Furthermore, our data provides evidence of vascular dysfunction in metastatic OS indicated by increased expression of VEGFA, an increased ANGPT2:ANGPT1 gene expression ratio, and decreased expression of SELE, the gene encoding the adhesion molecule E-selectin. Moreover, correlation analyses show an inverse relationship between lymphocyte abundance and markers of vascular dysfunction exclusively in the metastatic specimens. Together, our data shows that the non-metastatic OS specimens demonstrate increased expression of various immunotherapeutic targets in comparison metastatic specimens and identifies vascular dysfunction and lymphocyte exclusion as important processes for therapeutic intervention in metastatic disease.

Core Metabolism Shifts during Growth on Methanol versus Methane in the Methanotroph Methylomicrobium buryatense 5GB1.

Methylomicrobium buryatense 5GB1 is an obligate methylotroph which grows on methane or methanol with similar growth rates. It has long been assumed that the core metabolic pathways must be similar on the two substrates, but recent studies of methane metabolism in this bacterium suggest that growth on methanol might have significant differences from growth on methane. In this study, both a targeted metabolomics approach and a 13C tracer approach were taken to understand core carbon metabolism in M. buryatense 5GB1 during growth on methanol and to determine whether such differences occur. Our results suggest a systematic shift of active core metabolism in which increased flux occurred through both the Entner-Doudoroff (ED) pathway and the partial serine cycle, while the tricarboxylic acid (TCA) cycle was incomplete, in contrast to growth on methane. Using the experimental results as constraints, we applied flux balance analysis to determine the metabolic flux phenotype of M. buryatense 5GB1 growing on methanol, and the results are consistent with predictions based on ATP and NADH changes. Transcriptomics analysis suggested that the changes in fluxes and metabolite levels represented results of posttranscriptional regulation. The combination of flux balance analysis of the genome-scale model and the flux ratio from 13C data changed the solution space for a better prediction of cell behavior and demonstrated the significant differences in physiology between growth on methane and growth on methanol.IMPORTANCE One-carbon compounds such as methane and methanol are of increasing interest as sustainable substrates for biological production of fuels and industrial chemicals. The bacteria that carry out these conversions have been studied for many decades, but gaps exist in our knowledge of their metabolic pathways. One such gap is the difference between growth on methane and growth on methanol. Understanding such metabolism is important, since each has advantages and disadvantages as a feedstock for production of chemicals and fuels. The significance of our research is in the demonstration that the metabolic network is substantially altered in each case and in the delineation of these changes. The resulting new insights into the core metabolism of this bacterium now provide an improved basis for future strain design.

A Spatio-Temporal Pattern and Socio-Economic Factors Analysis of Improved Sanitation in China, 2006⁻2015.

Ensuring an adequate and safe access to sanitation is essential to prevent diseases. Using provincial spatial panel data reported in the China Health Statistical Yearbook and the China Statistical Yearbook, this paper analyzed the spatio-temporal characteristics of improved rural sanitation in 30 Chinese provinces during the period 2006⁻2015, and analyzed factors that may affect improved sanitation rates in rural China. Spatial autocorrelations of improved sanitation rates were computed via Global and Local Moran's I firstly, and then, inter-provincial disparities of improved sanitation were assessed by using the Theil index estimator; finally, the spatial panel model was employed to examine the potential socio-economic factors. Spatial autocorrelations results suggested that the provincial improved sanitation rates changes affect both the provinces themselves and the adjacent regions; Analysis of the spatial panel model revealed that factors such as GDP per capita, investment proportion ratio, centralized water supply, rural residents' expenditure were positively associated with improved sanitation rates, and illiteracy rate of people older than 15 was negatively related with improved sanitation rates. Socio-economic factors had affected the improved sanitation rates in 30 provinces in rural China. Thus, a series of policies, socio-economic measures and personal latrine literacy education should be given to improve the status of improved sanitation rates in rural China.

A pathway for biological methane production using bacterial iron-only nitrogenase.

Methane (CH4) is a potent greenhouse gas that is released from fossil fuels and is also produced by microbial activity, with at least one billion tonnes of CH4 being formed and consumed by microorganisms in a single year 1 . Complex methanogenesis pathways used by archaea are the main route for bioconversion of carbon dioxide (CO2) to CH4 in nature2-4. Here, we report that wild-type iron-iron (Fe-only) nitrogenase from the bacterium Rhodopseudomonas palustris reduces CO2 simultaneously with nitrogen gas (N2) and protons to yield CH4, ammonia (NH3) and hydrogen gas (H2) in a single enzymatic step. The amount of CH4 produced by purified Fe-only nitrogenase was low compared to its other products, but CH4 production by this enzyme in R. palustris was sufficient to support the growth of an obligate CH4-utilizing Methylomonas strain when the two microorganisms were grown in co-culture, with oxygen (O2) added at intervals. Other nitrogen-fixing bacteria that we tested also formed CH4 when expressing Fe-only nitrogenase, suggesting that this is a general property of this enzyme. The genomes of 9% of diverse nitrogen-fixing microorganisms from a range of environments encode Fe-only nitrogenase. Our data suggest that active Fe-only nitrogenase, present in diverse microorganisms, contributes CH4 that could shape microbial community interactions.

Oxygen-limited metabolism in the methanotroph Methylomicrobium buryatense 5GB1C.

The bacteria that grow on methane aerobically (methanotrophs) support populations of non-methanotrophs in the natural environment by excreting methane-derived carbon. One group of excreted compounds are short-chain organic acids, generated in highest abundance when cultures are grown under O2-starvation. We examined this O2-starvation condition in the methanotroph Methylomicrobium buryatense 5GB1. The M. buryatense 5GB1 genome contains homologs for all enzymes necessary for a fermentative metabolism, and we hypothesize that a metabolic switch to fermentation can be induced by low-O2 conditions. Under prolonged O2-starvation in a closed vial, this methanotroph increases the amount of acetate excreted about 10-fold, but the formate, lactate, and succinate excreted do not respond to this culture condition. In bioreactor cultures, the amount of each excreted product is similar across a range of growth rates and limiting substrates, including O2-limitation. A set of mutants were generated in genes predicted to be involved in generating or regulating excretion of these compounds and tested for growth defects, and changes in excretion products. The phenotypes and associated metabolic flux modeling suggested that in M. buryatense 5GB1, formate and acetate are excreted in response to redox imbalance. Our results indicate that even under O2-starvation conditions, M. buryatense 5GB1 maintains a metabolic state representing a combination of fermentation and respiration metabolism.

The oxidative TCA cycle operates during methanotrophic growth of the Type I methanotroph Methylomicrobium buryatense 5GB1.

Methanotrophs are a group of bacteria that use methane as sole carbon and energy source. Type I methanotrophs are gamma-proteobacterial methanotrophs using the ribulose monophosphate cycle (RuMP) cycle for methane assimilation. In order to facilitate metabolic engineering in the industrially promising Type I methanotroph Methylomicrobium buryatense 5GB1, flux analysis of cellular metabolism is needed and 13C tracer analysis is a foundational tool for such work. This biological system has a single-carbon input and a special network topology that together pose challenges to the current well-established methodology for 13C tracer analysis using a multi-carbon input such as glucose, and to date, no 13C tracer analysis of flux in a Type I methanotroph has been reported. In this study, we showed that by monitoring labeling patterns of several key intermediate metabolites in core metabolism, it is possible to quantitate the relative flux ratios for important branch points, such as the malate node. In addition, it is possible to assess the operation of the TCA cycle, which has been thought to be incomplete in Type I methanotrophs. Surprisingly, our analysis provides direct evidence of a complete, oxidative TCA cycle operating in M. buryatense 5GB1 using methane as sole carbon and energy substrate, contributing about 45% of the total flux for de novo malate production. Combined with mutant analysis, this method was able to identify fumA (METBUDRAFT_1453/MBURv2__60244) as the primary fumarase involved in the oxidative TCA cycle, among 2 predicted fumarases, supported by 13C tracer analysis on both fumA and fumC single knockouts. Interrupting the oxidative TCA cycle leads to a severe growth defect, suggesting that the oxidative TCA cycle functions to not only provide precursors for de novo biomass synthesis, but also to provide reducing power to the system. This information provides new opportunities for metabolic engineering of M. buryatense for the production of industrially relevant products.

Quorum Sensing in a Methane-Oxidizing Bacterium.

Aerobic methanotrophic bacteria use methane as their sole source of carbon and energy and serve as a major sink for the potent greenhouse gas methane in freshwater ecosystems. Dissecting the molecular details of how these organisms interact in the environment may increase our understanding of how they perform this important ecological role. Many bacterial species use quorum sensing (QS) systems to regulate gene expression in a cell density-dependent manner. We have identified a QS system in the genome of Methylobacter tundripaludum, a dominant methane oxidizer in methane enrichments of sediment from Lake Washington (Seattle, WA). We determined that M. tundripaludum produces primarily N-3-hydroxydecanoyl-l-homoserine lactone (3-OH-C10-HSL) and that its production is governed by a positive feedback loop. We then further characterized this system by determining which genes are regulated by QS in this methane oxidizer using transcriptome sequencing (RNA-seq) and discovered that this system regulates the expression of a putative nonribosomal peptide synthetase biosynthetic gene cluster. Finally, we detected an extracellular factor that is produced by M. tundripaludum in a QS-dependent manner. These results identify and characterize a mode of cellular communication in an aerobic methane-oxidizing bacterium.IMPORTANCE Aerobic methanotrophs are critical for sequestering carbon from the potent greenhouse gas methane in the environment, yet the mechanistic details of chemical interactions in methane-oxidizing bacterial communities are not well understood. Understanding these interactions is important in order to maintain, and potentially optimize, the functional potential of the bacteria that perform this vital ecosystem function. In this work, we identify a quorum sensing system in the aerobic methanotroph Methylobacter tundripaludum and use both chemical and genetic methods to characterize this system at the molecular level.