Following the Birth, Life, and Death of mRNAs in Single Cells.

Recent advances in single-molecule imaging of mRNAs in fixed and living cells have enabled the lives of mRNAs to be studied with unprecedented spatial and temporal detail. These approaches have moved beyond simply being able to observe specific events and have begun to allow an understanding of how regulation is coupled between steps in the mRNA life cycle. Additionally, these methodologies are now being applied in multicellular systems and animals to provide more nuanced insights into the physiological regulation of RNA metabolism.

Dopaminergic systems create reward seeking despite adverse consequences.

Resource-seeking behaviours are ordinarily constrained by physiological needs and threats of danger, and the loss of these controls is associated with pathological reward seeking1. Although dysfunction of the dopaminergic valuation system of the brain is known to contribute towards unconstrained reward seeking2,3, the underlying reasons for this behaviour are unclear. Here we describe dopaminergic neural mechanisms that produce reward seeking despite adverse consequences in Drosophila melanogaster. Odours paired with optogenetic activation of a defined subset of reward-encoding dopaminergic neurons become cues that starved flies seek while neglecting food and enduring electric shock punishment. Unconstrained seeking of reward is not observed after learning with sugar or synthetic engagement of other dopaminergic neuron populations. Antagonism between reward-encoding and punishment-encoding dopaminergic neurons accounts for the perseverance of reward seeking despite punishment, whereas synthetic engagement of the reward-encoding dopaminergic neurons also impairs the ordinary need-dependent dopaminergic valuation of available food. Connectome analyses reveal that the population of reward-encoding dopaminergic neurons receives highly heterogeneous input, consistent with parallel representation of diverse rewards, and recordings demonstrate state-specific gating and satiety-related signals. We propose that a similar dopaminergic valuation system dysfunction is likely to contribute to maladaptive seeking of rewards by mammals.

Inequities in sleep duration and quality among adolescents in Canada.

BACKGROUND: Several recent global events may have impacted adolescent sleep and exacerbated pre-existing disparities by social positions (i.e., social roles, identity or sociodemographic factors, and/or group memberships that are associated with power and oppression due to the structures and processes in a given society at given time). Current understanding of sleep among adolescents is critical to inform interventions for a more equitable future, given the short and long-term consequences of inadequate sleep on health and well-being. This study aimed to provide contemporary evidence on sleep disparities by key social positions among adolescents in Canada. METHODS: Cross-sectional analyses were conducted using self-reported data collected during 2020-2021 (the first full school year after the COVID-19 pandemic onset) from 52,138 students (mean [SD] age = 14.9 [1.5]) attending 133 Canadian secondary schools. Multiple regression models were used to test whether sleep quality (how well students slept during past week), duration (weekday, weekend, weighted daily average), and guideline adherence (8-10 h/day) differed by sex and gender, race and ethnicity, and socioeconomic status (SES). RESULTS: Females reported a mean [95% CI] difference of -1.7 [-3.7, 0.4] min/day less sleep on weekdays than males, but 7.1 [4.5, 9.6] min/day more sleep on weekends, resulting in no difference in average daily sleep between males and females. Females were less likely to report good quality sleep compared to males (AOR = 0.57 [0.54, 0.60]). SES followed a generally monotonic trend where higher scores were associated with more sleep on weekdays (Δhighest: lowest = -28.6 [-39.5, -17.6]) and weekends (Δhighest: lowest = -17.5 [-3.8, -31.2]) and greater likelihood of higher sleep quality (AORhighest: lowest = 3.04 [2.35, 3.92]). Relative to White adolescents, weekday and average daily sleep duration were lower among all other racial identities; mean differences ranged from ∼ 5-15 min/day, with Black students reporting the least sleep. CONCLUSIONS: Differences in sleep duration and quality were most profound among adolescents from the lowest and highest SES. Racial disparities were more evident on weekdays. Compensatory weekend sleep appears more pronounced in females than males. Addressing sleep inequities is critical, as a robust predictor of multiple health outcomes.

Exploring community insights on antimicrobial resistance in Nepal: a formative qualitative study.

BACKGROUND: Antimicrobial resistance (AMR) is the process by which microbes evolve mechanisms to survive the medicines designed to destroy them i.e. antimicrobials (AMs). Despite being a natural process, AMR is being hastened by the abuse of AMs. In context of Nepal, there is limited information on drivers of AMR and barriers in addressing it from a community perspective. This study explores the local language and terminology used around AMs in the community, commonly used AMs and reasons for their usage, how these AMs are sourced, and the perceived barriers to addressing AMR via One Health approach. METHODS: A phenomenological study design was utilized with applied qualitative research theoretically framed as pragmatism. Twelve in-depth interviews and informal discussions with a One Health focus, were purposively conducted with wide range of stakeholders and community resident of Kapilvastu municipality of Nepal during April 2022. The acquired data was analyzed manually via a thematic framework approach. The study obtained ethical approval from ethical review board of Nepal Health Research Council and University of Leeds. RESULTS: Nepali and Awadhi languages does not have specific words for AMs or AMR, which is understandable by the community people. Rather, community use full explanatory sentences. People use AMs but have incomplete knowledge about them and they have their own local words for these medicines. The knowledge and usage of AMs across human and animal health is impacted by socio-structural factors, limited Government regulation, inadequate supply of AMs in local government health facilities and the presence of various unregulated health providers that co-exist within the health system. Novel ideas such as the use of visual and smart technology, for instance mobile phones and social media exposure, can enable access to information about AMs and AMR. CONCLUSION: This study shows that terminology that is understandable by the community referring to AMs and AMR in Nepali and Awadhi languages does not exist, but full explanatory sentences and colloquial names are used. Despite regular utilisation, communities have incomplete knowledge regarding AMs. Since, knowledge alone cannot improve behaviour, behavioural interventions are required to address AMR via community engagement to co-produce their own solutions. TRIAL REGISTRATION: Not applicable.

Novel Insights on Obligate Symbiont Lifestyle and Adaptation to Chemosynthetic Environment as Revealed by the Giant Tubeworm Genome.

The mutualism between the giant tubeworm Riftia pachyptila and its endosymbiont Candidatus Endoriftia persephone has been extensively researched over the past 40 years. However, the lack of the host whole-genome information has impeded the full comprehension of the genotype/phenotype interface in Riftia. Here, we described the high-quality draft genome of Riftia, its complete mitogenome, and tissue-specific transcriptomic data. The Riftia genome presents signs of reductive evolution, with gene family contractions exceeding expansions. Expanded gene families are related to sulfur metabolism, detoxification, antioxidative stress, oxygen transport, immune system, and lysosomal digestion, reflecting evolutionary adaptations to the vent environment and endosymbiosis. Despite the derived body plan, the developmental gene repertoire in the gutless tubeworm is extremely conserved with the presence of a near intact and complete Hox cluster. Gene expression analyses establish that the trophosome is a multifunctional organ marked by intracellular digestion of endosymbionts, storage of excretory products, and hematopoietic functions. Overall, the plume and gonad tissues both in contact to the environment harbor highly expressed genes involved with cell cycle, programed cell death, and immunity indicating a high cell turnover and defense mechanisms against pathogens. We posit that the innate immune system plays a more prominent role into the establishment of the symbiosis during the infection in the larval stage, rather than maintaining the symbiostasis in the trophosome. This genome bridges four decades of physiological research in Riftia, whereas it simultaneously provides new insights into the development, whole organism functions, and evolution in the giant tubeworm.

E-CPR in Cardiac Arrest due to Accidental Hypothermia Using Intensivist Cannulators: A Case Series of Nine Consecutive Patients.

Background: Severe accidental hypothermia (AH) accounts for over 1300 deaths/year in the United States. Early extracorporeal life support (ECLS) is recommended for hypothermic cardiac arrest. We describe the use of a rapid-deployment extracorporeal cardiopulmonary resuscitation (E-CPR) team using intensivist physicians (IPs) as cannulators and report the outcomes of consecutive patients cannulated for ECLS to manage cardiac arrest due to AH. Methods: We reviewed all patients managed with veno-arterial (V-A) ECLS for hypothermic cardiac arrest between January 1, 2017 and November 1, 2021. For each patient- age, sex, cause of hypothermia, initial core temperature, initial rhythm, time from arrest to cannulation, cannula configuration, pH, lactate, potassium, cannulation complications, duration of ECLS, hospital length of stay, mortality, and cerebral performance category (CPC) at discharge were reviewed. Results: Nine consecutive patients were identified that underwent V-A ECLS for cardiac arrest due to AH. Seven (78%) were witnessed arrests. Initial rhythm was ventricular fibrillation (VF) in eight patients and pulseless electrical activity (PEA) in one. The mean initial core temperature was 23.8 degrees Celsius. The mean time from arrest to cannulation was 58 min (range 17 to 251 min). There were no complications related to cannulation. The mean duration of ECLS was 39.1 h. All nine patients were discharged alive with a Cerebral Performance score of one or two. Conclusion: In this case series of consecutive patients reporting intensivist-deployed E-CPR for cardiac arrest due to AH, all patients survived to discharge with a favorable neurologic outcome. A rapidly available E-CPR team utilizing intensivist cannulators may improve outcomes in patients with cardiac arrest due to AH.

Rapid implementation of an emergency on-site CKRT dialysate production system during the COVID-19 pandemic.

BACKGROUND: On December 29, 2021, during the delta wave of the Coronavirus Disease 2019 (COVID-19) pandemic, the stock of premanufactured solutions used for continuous kidney replacement therapy (CKRT) at the University of New Mexico Hospital (UNMH) was nearly exhausted with no resupply anticipated due to supply chain disruptions. Within hours, a backup plan, devised and tested 18 months prior, to locally produce CKRT dialysate was implemented. This report describes the emergency implementation and outcomes of this on-site CKRT dialysate production system. METHODS: This is a single-center retrospective case series and narrative report describing and reporting the outcomes of the implementation of an on-site CKRT dialysate production system. All adults treated with locally produced CKRT dialysate in December 2021 and January 2022 at UNMH were included. CKRT dialysate was produced locally using intermittent hemodialysis machines, hemodialysis concentrate, sterile parenteral nutrition bags, and connectors made of 3-D printed biocompatible rigid material. Outcomes analyzed included dialysate testing for composition and microbiologic contamination, CKRT prescription components, patient mortality, sequential organ failure assessment (SOFA) scores, and catheter-associated bloodstream infections (CLABSIs). RESULTS: Over 13 days, 22 patients were treated with 3,645 L of locally produced dialysate with a mean dose of 20.0 mL/kg/h. Fluid sample testing at 48 h revealed appropriate electrolyte composition and endotoxin levels and bacterial colony counts at or below the lower limit of detection. No CLABSIs occurred within 7 days of exposure to locally produced dialysate. In-hospital mortality was 81.8% and 28-day mortality was 68.2%, though illness severity was high, with a mean SOFA score of 14.5. CONCLUSIONS: Though producing CKRT fluid with IHD machines is not novel, this report represents the first description of the rapid and successful implementation of a backup plan for local CKRT dialysate production at a large academic medical center in the U.S. during the COVID-19 pandemic. Though conclusions are limited by the retrospective design and limited sample size of our analysis, our experience could serve as a guide for other centers navigating similar severe supply constraints in the future.

The influence of continuous renal replacement therapy on 1,3-ß-d-glucan levels in critically ill patients: a single-center retrospective propensity score study.

1,3-ß-d-Glucan (BDG) is commonly used for diagnosing invasive fungal infections (IFIs). While exposure to cellulose-based hemodialyzers is known to cause false-positive BDG results, the impact of modern hemofilters used in continuous renal replacement therapy (CRRT) remains unclear. This retrospective, single-center cohort study aimed to evaluate the effect of CRRT on BDG levels in critically ill patients. We included adult intensive care unit (ICU) patients with ≥1 BDG measurement between December 2019 and December 2020. The primary outcome was the rate of false-positive BDG results in patients exposed to CRRT compared to unexposed patients. Propensity score analysis was performed to control for confounding factors. A total of 103 ICU patients with ≥1 BDG level were identified. Most (72.8%) were medical ICU patients. Forty patients underwent CRRT using hemofilter membranes composed of sodium methallyl sulfonate copolymer (AN 69 HF) (82.5%) and of polyarylethersulfone (PAES) (17.5%). Among the 91 patients without proven IFI, 31 (34.1%) had false-positive BDG results. Univariable analysis showed an association between CRRT exposure and false-positive BDG results. However, the association between CRRT exposure and false-positive BDG results was no longer significant across three propensity score models employed: 1:1 match (n = 32) (odds ratio (OR) 1.65, p = .48), model-adjusted (n = 91) (OR 1.75, p = .38), quintile-adjusted (n = 91) (OR 1.78, p = .36). In this single-center retrospective analysis, exposure to synthetic CRRT membranes did not independently increase the risk of false-positive BDG results. Larger prospective studies are needed to further evaluate the association between CRRT exposure and false-positive BDG results in critically ill patients with suspected IFI.

Creating a framework to align antimicrobial resistance (AMR) research with the global guidance: a viewpoint.

We describe here an initial analysis of national and international guidance documents on antimicrobial resistance (AMR) to propose a framework to align AMR research activities with global guidance. The framework provides a summary roadmap for core activities in AMR research and highlights the need for interdisciplinary and One Health collaboration. This analysis also revealed limitations in the current guidance, including a lack of explicit mention of some research activities highly relevant to AMR and a dearth of concrete objectives; consequently, an over-reliance on global guidance could be funnelling research efforts down a generic trajectory without regard to contextual factors. We suggest this framework be used by academics and policymakers to align AMR research and guidance. However, we recommend that deeper exploration be undertaken to fully contextualize the development of meaningful questions based on current knowledge, methodologies and gap analyses.

Balanced Crystalloid Versus Normal Saline as Resuscitative Fluid in Diabetic Ketoacidosis.

BACKGROUND: Large volume resuscitation with normal saline (NS) may be associated with iatrogenic hyperchloremia and renal injury. OBJECTIVE: The purpose of this study was to assess clinical outcomes associated with the use of Lactated Ringer's (LR) compared to NS as resuscitative fluid in diabetic ketoacidosis (DKA). METHODS: Single-center, retrospective analysis of patients admitted for DKA. The primary objective of this study was to evaluate the incidence of iatrogenic hyperchloremia associated with fluid resuscitation using balanced crystalloid compared to NS. RESULTS: Iatrogenic hyperchloremia occurred more frequently in the NS group compared to the LR group (74.4% vs 64.2%; P = 0.05). Mean maximum serum chloride was higher in the NS group (115.7 mmol/L vs 113.7 mmol/L; P = 0.004). Incidence of hypernatremia was higher in the NS group (18.3% vs 9.3%; P = 0.02). There was no significant difference in the incidence of AKI; however, mean change in serum creatinine at 48 hours showed a significantly greater decrease in the LR group (-0.15 mg/dL vs -0.04 mg/dL; P = 0.002). No significant differences were found in intensive care unit (ICU) length of stay or total hospital length of stay. CONCLUSION AND RELEVANCE: This study found a statistically significant reduction in the incidence of iatrogenic hyperchloremia with the use of LR compared to NS as fluid resuscitation in DKA. Serum creatinine was more improved in the LR group versus NS group at 48 hours. Preferential use of balanced crystalloid for fluid resuscitation in DKA may reduce incidence of hyperchloremia and support renal recovery in this population.

Inequalities in Life Expectancy Across North Carolina: A Spatial Analysis of the Social Determinants of Health and the Index of Concentration at Extremes.

Health inequalities are characterized by spatial patterns of social, economic, and political factors. Life expectancy (LE) is a commonly used indicator of overall population health and health inequalities that allows for comparison across different spatial and temporal regions. The objective of this study was to examine geographic inequalities in LE across North Carolina census tracts by comparing the performance of 2 popular geospatial health indices: Social Determinants of Health (SDoH) and the Index of Concentration at Extremes (ICE). A principal components analysis (PCA) was used to address multicollinearity among variables and aggregate data into components to examine SDoH, while the ICE was constructed using the simple subtraction of geospatial variables. Spatial regression models were employed to compare both indices in relation to LE to evaluate their predictability for population health. For individual SDoH and ICE components, poverty and income had the strongest positive correlation with LE. However, the common spatial techniques of adding PCA components together for a final SDoH aggregate measure resulted in a poor relationship with LE. Results indicated that both metrics can be used to determine spatial patterns of inequities in LE and that the ICE metric has similar success to the more computationally complex SDoH metric. Public health practitioners may find the ICE metric's high predictability matched with lower data requirements to be more feasible to implement in population health monitoring.

Single-molecule mRNA and translation imaging in neurons.

Memory-relevant neuronal plasticity is believed to require local translation of new proteins at synapses. Understanding this process has necessitated the development of tools to visualize mRNA within relevant neuronal compartments. In this review, we summarize the technical developments that now enable mRNA transcripts and their translation to be visualized at single-molecule resolution in both fixed and live cells. These tools include single-molecule fluorescence in situ hybridization (smFISH) to visualize mRNA in fixed cells, MS2/PP7 labelling for live mRNA imaging and SunTag labelling to observe the emergence of nascent polypeptides from a single translating mRNA. The application of these tools in cultured neurons and more recently in whole brains promises to revolutionize our understanding of local translation in the neuronal plasticity that underlies behavioural change.

Cooccurring Activities of Two Autotrophic Pathways in Symbionts of the Hydrothermal Vent Tubeworm Riftia pachyptila.

Genome and proteome data predict the presence of both the reductive citric acid cycle (rCAC; also called the reductive tricarboxylic acid cycle) and the Calvin-Benson-Bassham cycle (CBB) in "Candidatus Endoriftia persephonae," the autotrophic sulfur-oxidizing bacterial endosymbiont from the giant hydrothermal vent tubeworm Riftia pachyptila. We tested whether these cycles were differentially induced by sulfide supply, since the synthesis of biosynthetic intermediates by the rCAC is less energetically expensive than that by the CBB. R. pachyptila was incubated under in situ conditions in high-pressure aquaria under low (28 to 40 µmol · h-1) or high (180 to 276 µmol · h-1) rates of sulfide supply. Symbiont-bearing trophosome samples excised from R. pachyptila maintained under the two conditions were capable of similar rates of CO2 fixation. Activities of the rCAC enzyme ATP-dependent citrate lyase (ACL) and the CBB enzyme 1,3-bisphosphate carboxylase/oxygenase (RubisCO) did not differ between the two conditions, although transcript abundances for ATP-dependent citrate lyase were 4- to 5-fold higher under low-sulfide conditions. δ13C values of internal dissolved inorganic carbon (DIC) pools were varied and did not correlate with sulfide supply rate. In samples taken from freshly collected R. pachyptila, δ13C values of lipids fell between those collected for organisms using either the rCAC or the CBB exclusively. These observations are consistent with cooccurring activities of the rCAC and the CBB in this symbiosis. IMPORTANCE Previous to this study, the activities of the rCAC and CBB in R. pachyptila had largely been inferred from "omics" studies of R. pachyptila without direct assessment of in situ conditions prior to collection. In this study, R. pachyptila was maintained and monitored in high-pressure aquaria prior to measuring its CO2 fixation parameters. Results suggest that ranges in sulfide concentrations similar to those experienced in situ do not exert a strong influence on the relative activities of the rCAC and the CBB. This observation highlights the importance of further study of this symbiosis and other organisms with multiple CO2-fixing pathways, which recent genomics and biochemical studies suggest are likely to be more prevalent than anticipated.

Analysis of serum HE4 levels in various histologic subtypes of epithelial ovarian cancer and other malignant tumors.

BACKGROUND: The measurement of serum HE4 levels has emerged as a sensitive and specific biomarker for epithelial ovarian cancers (EOCs). However, serum levels in women diagnosed with various histologic subtypes of EOC and in women with metastatic non-ovarian primary malignancies have not been widely reported. OBJECTIVE: The goal of this study was to identify how serum HE4 levels vary in women diagnosed with different histologic subtypes of EOC and non-ovarian malignancies. METHODS: Data from six prospective pelvic mass clinical trials was combined and an evaluation of serum HE4 levels in women diagnosed with a malignancy was performed. For all patients, serum was obtained prior to surgery and final pathology, including primary tumor site, histologic subtype, grade and stage, were recorded. The mean, median, standard deviation, maximum, and minimum HE4 levels were determined for each group. RESULTS: A total of 984 patients were included in this study, with the average patient age being 60 years old. There were 230 premenopausal and 754 postmenopausal patients. Serum HE4 levels were elevated (≥70.0 pMol) in 85%of EOCs, 40%of LMP tumors, 21%of non-EOCs (germ cell tumors), 25%of cervical cancers, and 47%of non-gynecologic metastatic cancers. Analysis of histologic subtypes revealed 90%(n = 391) of serous, 85%(n = 73) of endometrioid, 45%(n = 42) of mucinous, 86%(n = 51) of mixed tumors, and 69%(n = 36) of clear cell tumors had elevated serum HE4 levels. CONCLUSIONS: Serum HE4 levels are most often elevated in women with high grade serous and endometrioid EOCs, and though serum elevations are seen more often with advanced stage disease, HE4 is also often elevated in early stage disease and lower grade tumors.

Nanoliposomal irinotecan (Nal-IRI)-based chemotherapy after irinotecan -based chemotherapy in patients with pancreas cancer.

BACKGROUND: Nanoliposomal irinotecan (Nal-IRI) is a preferred second-line treatment for metastatic pancreas cancer. It is unclear, however, whether patients who had received irinotecan derive benefit. METHODS: Medical records of metastatic pancreas cancer patients who had received irinotecan and then Nal-IRI were reviewed. The primary endpoint was overall survival after the initiation of Nal-IRI (an a priori threshold of >4 months defined success); adverse events and quotes from the medical record on decision-making were also recorded. RESULTS: Sixty four patients met eligibility criteria with a median age of 65 years (range: 36, 80 years). The median overall survival from initiation of Nal-IRI was 5.1 months (95% confidence interval (CI): 4.3, 5.6 months). An exploratory comparison, based on no cancer progression with irinotecan versus progression, showed improved survival with Nal-IRI in the former group: 6.1 months (95% CI: 5.1, 9.3 months) versus 4.3 months (95% CI: 2.3, 4.8 months); p = 0.0006. Nal-IRI adverse events occurred as expected. Qualitative data illustrate several themes, including "limited treatment options," which appeared to drive the decision to prescribe Nal-IRI. CONCLUSION: Nal-IRI might be considered in pancreas cancer patients who had received irinotecan, particularly in the absence of disease progression with the latter.

Bees prefer foods containing neonicotinoid pesticides.

The impact of neonicotinoid insecticides on insect pollinators is highly controversial. Sublethal concentrations alter the behaviour of social bees and reduce survival of entire colonies. However, critics argue that the reported negative effects only arise from neonicotinoid concentrations that are greater than those found in the nectar and pollen of pesticide-treated plants. Furthermore, it has been suggested that bees could choose to forage on other available flowers and hence avoid or dilute exposure. Here, using a two-choice feeding assay, we show that the honeybee, Apis mellifera, and the buff-tailed bumblebee, Bombus terrestris, do not avoid nectar-relevant concentrations of three of the most commonly used neonicotinoids, imidacloprid (IMD), thiamethoxam (TMX), and clothianidin (CLO), in food. Moreover, bees of both species prefer to eat more of sucrose solutions laced with IMD or TMX than sucrose alone. Stimulation with IMD, TMX and CLO neither elicited spiking responses from gustatory neurons in the bees' mouthparts, nor inhibited the responses of sucrose-sensitive neurons. Our data indicate that bees cannot taste neonicotinoids and are not repelled by them. Instead, bees preferred solutions containing IMD or TMX, even though the consumption of these pesticides caused them to eat less food overall. This work shows that bees cannot control their exposure to neonicotinoids in food and implies that treating flowering crops with IMD and TMX presents a sizeable hazard to foraging bees.

Hydrogen Does Not Appear To Be a Major Electron Donor for Symbiosis with the Deep-Sea Hydrothermal Vent Tubeworm Riftia pachyptila.

Use of hydrogen gas (H2) as an electron donor is common among free-living chemolithotrophic microorganisms. Given the presence of this dissolved gas at deep-sea hydrothermal vents, it has been suggested that it may also be a major electron donor for the free-living and symbiotic chemolithoautotrophic bacteria that are the primary producers at these sites. Giant Riftia pachyptila siboglinid tubeworms and their symbiotic bacteria ("Candidatus Endoriftia persephone") dominate many vents in the Eastern Pacific, and their use of sulfide as a major electron donor has been documented. Genes encoding hydrogenase are present in the "Ca Endoriftia persephone" genome, and proteome data suggest that these genes are expressed. In this study, high-pressure respirometry of intact R. pachyptila and incubations of trophosome homogenate were used to determine whether this symbiotic association could also use H2 as a major electron donor. Measured rates of H2 uptake by intact R. pachyptila in high-pressure respirometers were similar to rates measured in the absence of tubeworms. Oxygen uptake rates in the presence of H2 were always markedly lower than those measured in the presence of sulfide, as was the incorporation of 13C-labeled dissolved inorganic carbon. Carbon fixation by trophosome homogenate was not stimulated by H2, nor was hydrogenase activity detectable in these samples. Though genes encoding [NiFe] group 1e and [NiFe] group 3b hydrogenases are present in the genome and transcribed, it does not appear that H2 is a major electron donor for this system, and it may instead play a role in intracellular redox homeostasis.IMPORTANCE Despite the presence of hydrogenase genes, transcripts, and proteins in the "Ca Endoriftia persephone" genome, transcriptome, and proteome, it does not appear that R. pachyptila can use H2 as a major electron donor. For many uncultivable microorganisms, omic analyses are the basis for inferences about their activities in situ However, as is apparent from the study reported here, there are dangers in extrapolating from omics data to function, and it is essential, whenever possible, to verify functions predicted from omics data with physiological and biochemical measurements.

Single molecule fluorescence in situ hybridisation for quantitating post-transcriptional regulation in Drosophila brains.

RNA in situ hybridization is a powerful method to investigate post-transcriptional regulation, but analysis of intracellular mRNA distributions in thick, complex tissues like the brain poses significant challenges. Here, we describe the application of single-molecule fluorescent in situ hybridization (smFISH) to quantitate primary nascent transcription and post-transcriptional regulation in whole-mount Drosophila larval and adult brains. Combining immunofluorescence and smFISH probes for different regions of a single gene, i.e., exons, 3'UTR, and introns, we show examples of a gene that is regulated post-transcriptionally and one that is regulated at the level of transcription. Our simple and rapid protocol can be used to co-visualise a variety of different transcripts and proteins in neuronal stem cells as well as deep brain structures such as mushroom body neuropils, using conventional confocal microscopy. Finally, we introduce the use of smFISH as a sensitive alternative to immunofluorescence for labelling specific neural stem cell populations in the brain.