Early detection of necrosis in low-enhanced pancreatic parenchyma using contrast-enhanced computed tomography was a better predictor of clinical outcomes than pancreatic inflammation: A multicentric cohort study of severe acute pancreatitis.

OBJECTIVES: We aim to assess the early use of contrast-enhanced computed tomography (CECT) of patients with severe acute pancreatitis (SAP) using the computed tomography severity index (CTSI) in prognosis prediction. The CTSI combines quantification of pancreatic and extrapancreatic inflammation with the extent of pancreatic necrosis. METHODS: Post-hoc retrospective analysis of a large, multicentric database (44 institutions) of SAP patients in Japan. The area under the curve (AUC) of the CTSI for predicting mortality and the odds ratio (OR) of the extent of pancreatic inflammation and necrosis were calculated using multivariable analysis. RESULTS: In total, 1097 patients were included. The AUC of the CTSI for mortality was 0.65 (95 % confidence interval [CI:] [0.59-0.70]; p < 0.001). In multivariable analysis, necrosis 30-50 % and >50 % in low-enhanced pancreatic parenchyma (LEPP) was independently associated with a significant increase in mortality, with OR 2.04 and 95 % CI 1.01-4.12 (P < 0.05) and OR 3.88 and 95 % CI 2.04-7.40 (P < 0.001), respectively. However, the extent of pancreatic inflammation was not associated with mortality, regardless of severity. CONCLUSIONS: The degree of necrosis in LEPP assessed using early CECT of SAP was a better predictor of mortality than the extent of pancreatic inflammation.

Impact of Nutritional Therapy During Intensive Care Unit Admission on Post Intensive Care Syndrome in Patients COVID-19.

INTRODUCTION: Nutritional therapy is an important component of intensive care. We investigated the associations of nutritional therapy in the acute phase of severe COVID-19 with the long-term outcomes of post-intensive care syndrome (PICS) and post-COVID-19 conditions. METHODS: A questionnaire on the health status after COVID-19 was sent to patients 1 year after infection and PICS was evaluated. Total energy and protein intakes during the first week after admission to the intensive care unit (ICU) were calculated. The primary endpoint was a decrease in quality of life (QOL) defined by EuroQol5-dimensions 5-level (EQ5D5L) <0.8. A multivariable regression analysis was used to examine. RESULTS: A total of 220 ICU patients were included in this study. Median total energy and protein intakes were 65.1 kcal/kg/week and 3.3 g/kg/week, respectively. Total energy and protein intakes were associated with EQ5D5L scores (Energy: unit odds ratio 0.98 [0.97-0.99], p-value<0.01; protein: unit odds ratio 0.72 [0.59-0.87], p-value<0.01). Insufficient total energy and protein intakes were associated with malaise, arthralgia, myalgia, palpitations, sleep disturbance, and muscle weakness. CONCLUSIONS: Poor nutrition during the first week after ICU admission was associated with a decreased QOL one year after. These nutrition shortages were also associated with an increased risk of developing PICS, post-COVID-19 conditions, which may contribute to decreased QOL.

Relationship between critical care nutrition and post-intensive care syndrome in surviving ventilated patients with COVID-19: a multicenter prospective observational study.

The impact of nutrition therapy in the acute phase on post-intensive care syndrome (PICS) remains unclear. We conducted a multicenter prospective study on adult patients with COVID-19 who required mechanical ventilation for more than three days. The questionnaire was mailed after discharge. Physical PICS, defined as less than 90 points on the Barthel index (BI), was assigned as the primary outcome. We examined the types of nutrition therapy in the first week that affected PICS components. 269 eligible patients were evaluated 10 months after discharge. Supplemental parenteral nutrition (SPN) >400 kcal/day correlated with a lower occurrence of physical PICS (10% vs 21.92%, p = 0.042), whereas the amounts of energy and protein provided, early enteral nutrition, and a gradual increase in nutrition delivery did not, and none correlated with cognitive or mental PICS. A multivariable regression analysis revealed that SPN had an independent impact on physical PICS (odds ratio 0.33, 95% CI 0.12-0.92, p = 0.034), even after adjustments for age, sex, body mass index and severity. Protein provision ≥1.2 g/kg/day was associated with a lower occurrence of physical PICS (odds ratio 0.42, 95% CI 0.16-1.08, p = 0.071). In conclusion, SPN in the acute phase had a positive impact on physical PICS for ventilated patients with COVID-19.

Early Elevation of Cell-Free DNA After Acute Mesenteric Ischemia in Rats.

BACKGROUND: Acute mesenteric ischemia (AMI) is challenging to diagnose in the early phase. We tested the hypothesis that blood levels of cell-free DNA would increase early after AMI. In addition, proteome analysis was conducted as an exploratory analysis to identify other potential diagnostic biomarkers. METHODS: Mesenteric ischemia, abdominal sepsis, and sham model were compared in Sprague-Dawley rats. The abdominal sepsis model was induced by cecum puncture and mesenteric ischemia model by ligation of the superior mesenteric artery. Blood levels of cell-free DNA were measured 2 h and 6 h after wound closure. Shotgun proteome analysis was performed using plasma samples obtained at the 2 h timepoint; quantitative analysis was conducted for proteins detected exclusively in the AMI models. RESULTS: Blood cell-free DNA levels at 2 h after wound closure were significantly higher in the AMI model than in the sham and the abdominal sepsis models (P < 0.05). Cell-free DNA was positively correlated with the pathologic ischemia severity score (correlation coefficient 0.793-0.834, P < 0.001). Derivative proteome analysis in blood at 2-h time point revealed higher intensity of paraoxonase-1 in the AMI models than in the abdominal sepsis models; the significantly high blood paraoxonase-1 levels in the AMI models were confirmed in a separate quantitative analysis (P = 0.015). CONCLUSIONS: Cell-free DNA was demonstrated to be a promising biomarker for the early diagnosis of mesenteric ischemia in a rat model of AMI. Paraoxonase-1 may also play a role in the differential diagnosis of mesenteric ischemia from abdominal sepsis. The current results warrant further investigation in human studies.

Cell Lysis Directed by SulA in Response to DNA Damage in Escherichia coli.

The SOS response is induced upon DNA damage and the inhibition of Z ring formation by the product of the sulA gene, which is one of the LexA-regulated genes, allows time for repair of damaged DNA. On the other hand, severely DNA-damaged cells are eliminated from cell populations. Overexpression of sulA leads to cell lysis, suggesting SulA eliminates cells with unrepaired damaged DNA. Transcriptome analysis revealed that overexpression of sulA leads to up-regulation of numerous genes, including soxS. Deletion of soxS markedly reduced the extent of cell lysis by sulA overexpression and soxS overexpression alone led to cell lysis. Further experiments on the SoxS regulon suggested that LpxC is a main player downstream from SoxS. These findings suggested the SulA-dependent cell lysis (SDCL) cascade as follows: SulA→SoxS→LpxC. Other tests showed that the SDCL cascade pathway does not overlap with the apoptosis-like and mazEF cell death pathways.

Mechanical Insufflation-exsufflation for the Prevention of Ventilator-associated Pneumonia in Intensive Care Units: A Retrospective Cohort Study.

AIM: Ventilator-associated pneumonia (VAP) is the most common intensive care unit (ICU)-acquired infection. The current study aimed to assess the efficacy of mechanical insufflation-exsufflation (MI-E) in preventing VAP in critically ill patients. MATERIALS AND METHODS: This retrospective cohort study was conducted at the ICU of Chiba University Hospital between January 2014 and September 2017. The inclusion criteria were patients who required invasive mechanical ventilation ≥48 hours and those who underwent rehabilitation, including chest physical therapy (CPT). In 2015, the study institution started the use of MI-E in patients with impaired cough reflex. From January to December 2014, patients undergoing CPT were classified under the historical control group, and those who received treatment using MI-E from January 2015 to September 2017 were included in the intervention group. The patients received treatment using MI-E via the endotracheal or tracheostomy tube, with insufflation-exsufflation pressure of 15-40 cm H2O. The treatment frequency was one to three sessions daily, and a physical therapist who is experienced in using MI-E facilitated the treatment. RESULTS: From January 2015 to September 2017, 11 patients received treatment using MI-E. Of the 169 patients screened in 2014, 19 underwent CPT. The incidence of VAP was significantly different between the CPT and MI-E groups (84.2% [16/19] vs 26.4% [3/11], p = 0.011). After adjusting for covariates, a multivariate logistic regression analysis was performed, and results showed that the covariates were not associated with the incidence of VAP. CONCLUSION: This retrospective cohort study suggests that the use of MI-E in critically ill patients is independently associated with a reduced incidence of VAP. CLINICAL SIGNIFICANCE: Assessing the efficacy of MI-E to prevent VAP. HOW TO CITE THIS ARTICLE: Kuroiwa R, Tateishi Y, Oshima T, Inagaki T, Furukawa S, Takemura R, et al. Mechanical Insufflation-exsufflation for the Prevention of Ventilator-associated Pneumonia in Intensive Care Units: A Retrospective Cohort Study. Indian J Crit Care Med 2021;25(1):62-66.

Etiology and mortality in severe acute pancreatitis: A multicenter study in Japan.

BACKGROUND/OBJECTIVES: Severe acute pancreatitis (SAP) has a high mortality rate despite ongoing attempts to improve prognosis through a various therapeutic modalities. This study aimed to delineate etiology-based routes that may guide clinical decisions for the treatment of SAP. METHODS: Using data from a recent retrospective multicenter study in Japan, we analyzed the association between clinical outcomes, mainly in-hospital mortality and pancreatic infection, and various etiologies while considering confounding factors. We performed additional multivariate analyses and built decision tree models. RESULTS: The 1097 participating patients were classified into the following groups by etiology: alcohol (n = 436, 39.7%); cholelithiasis (n = 230, 21.0%); idiopathic (n = 227, 20.7%); and others (n = 204, 18.6%). Mortality at hospital discharge was 8.4%, 12.2%, 16.7%, and 16.2% in the alcohol, cholelithiasis, idiopathic, and others groups, respectively. According to multivariable analysis, early enteral nutrition (EN) was significantly associated with reduced in-hospital mortality only in the cholelithiasis group. However, there was a consistent association between age and the need for mechanical ventilation and increased mortality, regardless of etiology. Our decision tree models presented different contributing factors depending on the etiology and patient background. Interaction analysis showed that EN and the use of prophylactic antibiotics may influence these results differently according to etiology. CONCLUSIONS: No study has yet used comprehensive models to investigate etiology-related prognostic factors for SAP; our results can, therefore, be used as a reference for improving clinical decisions.

Significance of body temperature in elderly patients with sepsis.

BACKGROUND: Elderly patients have a blunted host response, which may influence vital signs and clinical outcomes of sepsis. This study was aimed to investigate whether the associations between the vital signs and mortality are different in elderly and non-elderly patients with sepsis. METHODS: This was a retrospective observational study. A Japanese multicenter sepsis cohort (FORECAST, n = 1148) was used for the discovery analyses. Significant discovery results were tested for replication using two validation cohorts of sepsis (JAAMSR, Japan, n = 624; SPH, Canada, n = 1004). Patients were categorized into elderly and non-elderly groups (age ≥ 75 or < 75 years). We tested for association between vital signs (body temperature [BT], heart rate, mean arterial pressure, systolic blood pressure, and respiratory rate) and 90-day in-hospital mortality (primary outcome). RESULTS: In the discovery cohort, non-elderly patients with BT < 36.0 °C had significantly increased 90-day mortality (P = 0.025, adjusted hazard ratio 1.70, 95% CI 1.07-2.71). In the validation cohorts, non-elderly patients with BT < 36.0 °C had significantly increased mortality (JAAMSR, P = 0.0024, adjusted hazard ratio 2.05, 95% CI 1.29-3.26; SPH, P = 0.029, adjusted hazard ratio 1.36, 95% CI 1.03-1.80). These differences were not observed in elderly patients in the three cohorts. Associations between the other four vital signs and mortality were not different in elderly and non-elderly patients. The interaction of age and hypothermia/fever was significant (P < 0.05). CONCLUSIONS: In septic patients, we found mortality in non-elderly sepsis patients was increased with hypothermia and decreased with fever. However, mortality in elderly patients was not associated with BT. These results illuminate the difference in the inflammatory response of the elderly compared to non-elderly sepsis patients.

Clinical course of a critically ill patient with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Although several studies have reported on the clinical and epidemiological characteristics of the patient with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), clinical course of the most severe cases requiring treatment in ICU have been insufficiently reported. A 73-year-old man traveling on a cruise ship with history of hypertension and dyslipidemia developed high fever, dyspnea and cough after 7 days of steroid treatment for sudden sensorineural hearing loss, and tested positive for SARS-CoV-2 in sputa polymerase chain reaction (PCR) examination. His respiratory function deteriorated despite treatments with lopinavir/ritonavir, oseltamivir, azithromycin and meropenem at a regional hospital. He was intubated and transferred to the ICU in the tertiary university hospital on day 10 (ICU day 1). Interferon beta-1b subcutaneous injection was initiated immediately to enhance anti-viral therapy, and favipiravir on ICU day 10 upon availability. Progression of organ dysfunctions necessitated inhalation of nitrogen oxide for respiratory dysfunction, noradrenaline for cardiovascular dysfunction and continuous renal replacement therapy for renal dysfunction. His blood samples PCR also tested positive for SARS-CoV-2, indicating viremia, concomitantly with elevated IL-6 levels. VV-ECMO was initiated after sudden exacerbation of respiratory dysfunction on ICU day 7 to maintain oxygenation. The sustained excessive inflammatory cytokines in the present case might have led to the exacerbation of the disease, requiring vigorous organ support therapies to allow for survival and recovery from the rapid progression of multiple organ dysfunctions and severe respiratory failure.

Large Volume Fluid Resuscitation for Severe Acute Pancreatitis is Associated With Reduced Mortality: A Multicenter Retrospective Study.

BACKGROUND AND AIMS: Although fluid resuscitation is critical in acute pancreatitis, the optimal fluid volume is unknown. The aim of this study is to evaluate the association between the volume of fluid administered and clinical outcomes in patients with severe acute pancreatitis (SAP). METHODS: We conducted a multicenter retrospective study at 44 institutions in Japan. Inclusion criteria were age 18 years or older, and diagnosed with SAP from 2009 to 2013. Patients were stratified into 2 groups: administered fluid volume <6000 and ≥6000 mL in the first 24 hours. We evaluated the association between the 2 groups and clinical outcomes using multivariable logistic regression analysis. The primary outcome was in-hospital mortality. Secondary outcomes included the incidence of pancreatic infection and the need for surgical intervention. RESULTS: We analyzed 1097 patients, and the mean fluid volume administered was 5618±3018 mL (mean±SD), with 708 and 389 patients stratified into the fluid <6000 mL and fluid ≥6000 mL groups, respectively. Overall in-hospital mortality was 12.3%. The fluid ≥6000 mL group had significantly higher mortality than the fluid <6000 mL group (univariable analysis, 15.9% vs. 10.3%; P<0.05). In multivariable logistic regression analysis, administration of ≥6000 mL of fluid within the first 24 hours was significantly associated with reduced mortality (odds ratio, 0.58; P<0.05). No significant association was found between the administered fluid volume and pancreatic infection, or between the volume administered and the need for surgical intervention. CONCLUSIONS: In patients with SAP, administration of a large fluid volume within the first 24 hours is associated with decreased mortality.

Association between serum levels of interleukin-6 on ICU admission and subsequent outcomes in critically ill patients with acute kidney injury.

BACKGROUND: Exacerbated inflammatory response is considered one of the key elements of acute kidney injury (AKI). Interleukin-6 (IL-6) is an inflammatory cytokine that plays important roles in the inflammatory response and may be useful for predicting the clinical outcomes in patients with AKI. However, supporting evidence adapted to the current KDIGO criteria is lacking. METHODS: AKI patients admitted to the ICU between Jan 2011 and Dec 2015 were retrospectively screened. Patients were assigned to three groups by admission IL-6 tertiles. Associations between IL-6 on ICU admission and in-hospital 90-day mortality, short-term/long-term renal function were analyzed. RESULTS: Patients (n = 646) were divided into low (1.5-150.2 pg/mL), middle (152.0-1168 pg/mL), and high (1189-2,346,310 pg/mL) IL-6 on ICU admission groups. Patients in the high IL-6 group had higher in-hospital 90-day mortality (low vs. middle vs. high, P = 0.0050), lower urine output (low vs. middle vs. high, P < 0.0001), and an increased probability of persistent of anuria for ≥12 h (low vs. middle vs. high, P < 0.0001) within 72 h after ICU admission. In contrast, the high IL-6 group had a lower incidence of persistent AKI at 90 days after the ICU admission in survivors (low vs. middle vs. high, P = 0.013). CONCLUSIONS: Serum levels of IL-6 on ICU admission may predict short-term renal function and mortality in AKI patients and were associated with renal recovery in survivors.

CO2 and O2 removal during continuous veno-venous hemofiltration: a pilot study.

BACKGROUND: Carbon dioxide (CO2) accumulation is a challenging issue in critically ill patients. CO2 can be eliminated by renal replacement therapy but studies are scarce and clinical relevance is unknown. We prospectively studied CO2 and O2 behavior at different sample points of continuous veno-venous hemofiltration (CVVH) and build a model to calculate CO2 removal bedside. METHODS: In 10 patients receiving standard CVVH under citrate anticoagulation, blood gas analysis was performed at different sample points within the CVVH circuit. Citrate was then replaced by NaCl 0.9% and sampling was repeated. Total CO2 (tCO2), CO2 flow (V̇CO2) and O2 flow (V̇O2) were compared between different sample points. The effect of citrate on transmembrane tCO2 was evaluated. Wilcoxon matched-pairs signed rank test was performed to evaluate significance of difference between 2 data sets. Friedman test was used when more data sets were compared. RESULTS: V̇CO2 in the effluent (26.0 ml/min) correlated significantly with transmembrane V̇CO2 (24.2 ml/min). This represents 14% of the average expired V̇CO2 in ventilated patients. Only 1.3 ml/min CO2 was removed in the de-aeration chamber, suggesting that CO2 was almost entirely cleared across the membrane filter. tCO2 values in effluent, before, and after the filter were not statistically different. Transmembrane tCO2 under citrate or NaCl 0.9% predilution also did not differ significantly. No changes in V̇O2 were observed throughout the CVVH circuit. Based on recorded data, formulas were constructed that allow bedside evaluation of CVVH-attributable CO2 removal. CONCLUSION: A relevant amount of CO2 is removed by CVVH and can be quantified by one simple blood gas analysis within the circuit. Future studies should assess the clinical impact of this observation. TRIAL REGISTRATION: The trial was registered at https://clinicaltrials.gov with trial registration number NCT03314363 on October 192,017.

Correction to: CO2 and O2 removal during continuous veno-venous hemofiltration: a pilot study.

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H-NS Facilitates Sequence Diversification of Horizontally Transferred DNAs during Their Integration in Host Chromosomes.

Bacteria can acquire new traits through horizontal gene transfer. Inappropriate expression of transferred genes, however, can disrupt the physiology of the host bacteria. To reduce this risk, Escherichia coli expresses the nucleoid-associated protein, H-NS, which preferentially binds to horizontally transferred genes to control their expression. Once expression is optimized, the horizontally transferred genes may actually contribute to E. coli survival in new habitats. Therefore, we investigated whether and how H-NS contributes to this optimization process. A comparison of H-NS binding profiles on common chromosomal segments of three E. coli strains belonging to different phylogenetic groups indicated that the positions of H-NS-bound regions have been conserved in E. coli strains. The sequences of the H-NS-bound regions appear to have diverged more so than H-NS-unbound regions only when H-NS-bound regions are located upstream or in coding regions of genes. Because these regions generally contain regulatory elements for gene expression, sequence divergence in these regions may be associated with alteration of gene expression. Indeed, nucleotide substitutions in H-NS-bound regions of the ybdO promoter and coding regions have diversified the potential for H-NS-independent negative regulation among E. coli strains. The ybdO expression in these strains was still negatively regulated by H-NS, which reduced the effect of H-NS-independent regulation under normal growth conditions. Hence, we propose that, during E. coli evolution, the conservation of H-NS binding sites resulted in the diversification of the regulation of horizontally transferred genes, which may have facilitated E. coli adaptation to new ecological niches.

Non-invasive monitoring using photoplethysmography technology.

We evaluated the accuracy and precision of a novel non-invasive monitoring device in comparison with conventional monitoring methods used in intensive care units (ICU). The study device was developed to measure blood pressure, pulse rate, respiratory rate, and oxygen saturation, continuously with a single sensor using the photoplethysmographic technique. Patients who were monitored with arterial pressure lines in the ICU were enrolled. Systolic and diastolic blood pressure, pulse rate, respiratory rate, and arterial oxygen saturation were measured continuously for 30 min at 5-min intervals using the conventional methods and the study device. The primary outcome variable was blood pressure. Blood pressure measured by the study device highly correlated with the arterial pressure line values (correlation coefficients > 0.95). Percent errors for systolic, diastolic and mean blood pressures were 2.4% and 6.7% and 6.5%, respectively. Percent errors for pulse rate, respiratory rate and oxygen saturation were 3.4%, 5.6% and 1.4%, respectively. The non-invasive, continuous, multi-parameter monitoring device presented high level of agreement with the invasive arterial blood pressure monitoring, along with sufficient accuracy and precision in the measurements of pulse rate, respiratory rate, and oxygen saturation.

Small RNA Esr41 inversely regulates expression of LEE and flagellar genes in enterohaemorrhagic Escherichia coli.

Enterohaemorrhagic Escherichia coli (EHEC) is a life-threatening human pathogen worldwide. The locus of enterocyte effacement (LEE) in EHEC encodes a type three secretion system and effector proteins, all of which are essential for bacterial adherence to host cells. When LEE expression is activated, flagellar gene expression is down-regulated because bacterial flagella induce the immune responses of host cells at the infection stage. Therefore, this inverse regulation is also important for EHEC infection. We report here that a small regulatory RNA (sRNA), Esr41, mediates LEE repression and flagellar gene activation. Multiple copies of esr41 abolished LEE expression by down-regulating the expression of ler and pch, which encode positive regulators of LEE. This regulation led to reduced EHEC adhesion to host cells. Translational gene-reporter fusion experiments revealed that Esr41 regulates ler expression at a post-transcriptional level, and pch transcription, probably via an unknown target of Esr41. Esr41-mediated ler and pch repression was not observed in cells lacking hfq, which encodes an RNA-binding protein essential for most sRNA functions, indicating that Esr41 acts in an Hfq-dependent manner. We previously reported an increase in cell motility induced by Esr41. This motility enhancement was also observed in EHEC lacking ler, showing that Esr41-mediated enhancement of cell motility is in a ler-independent manner. In addition, Esr41 activated the expression of flagellar Class 3 genes by indirectly inducing the transcription of fliA, which encodes the sigma factor for flagellar synthesis. These results suggest that Esr41 plays important roles in the inverse regulation of LEE and flagellar gene expression.

Enlargement of Deinococcus grandis spheroplasts requires Mg2+ or Ca2.

While the cell wall strictly controls cell size and morphology in bacteria, spheroplasts lack cell walls and can become enlarged in growth medium under optimal conditions. Optimal conditions depend on the bacterial species. We frequently observed extreme enlargement of spheroplasts of the radiation-resistant bacterium Deinococcus grandis in Difco Marine Broth 2216, but not in TGY broth (a commonly used growth medium for Deinococcus). Thorough investigation of media components showed that the presence of Mg2+ or Ca2+ promoted extreme spheroplast enlargement, synthesizing the outer membrane. Our findings strongly suggest that Mg2+ or Ca2+ enlarges spheroplasts, which could change the lipid composition of the spheroplast membrane.

Innovations in energy expenditure assessment.

PURPOSE OF REVIEW: Optimal nutritional therapy has been associated with better clinical outcomes and requires providing energy as closed as possible to measured energy expenditure. We reviewed the current innovations in energy expenditure assessment in humans, focusing on indirect calorimetry and other new alternative methods. RECENT FINDINGS: Although considered the reference method to measure energy expenditure, the use of indirect calorimetry is currently limited by the lack of an adequate device. However, recent technical developments may allow a broader use of indirect calorimetry for in-patients and out-patients. An ongoing international academic initiative to develop a new indirect calorimeter aimed to provide innovative and affordable technical solutions for many of the current limitations of indirect calorimetry. New alternative methods to indirect calorimetry, including CO2 measurements in mechanically ventilated patients, isotopic approaches and accelerometry-based fitness equipments, show promises but have been either poorly studied and/or are not accurate compared to indirect calorimetry. Therefore, to date, energy expenditure measured by indirect calorimetry remains the gold standard to guide nutritional therapy. SUMMARY: Some new innovative methods are demonstrating promises in energy expenditure assessment, but still need to be validated. There is an ongoing need for easy-to-use, accurate and affordable indirect calorimeter for daily use in in-patients and out-patients.

Genome-Wide Analysis of ResD, NsrR, and Fur Binding in Bacillus subtilis during Anaerobic Fermentative Growth by In Vivo Footprinting.

Upon oxygen limitation, the Bacillus subtilis ResE sensor kinase and its cognate ResD response regulator play primary roles in the transcriptional activation of genes functioning in anaerobic respiration. The nitric oxide (NO)-sensitive NsrR repressor controls transcription to support nitrate respiration. In addition, the ferric uptake repressor (Fur) can modulate transcription under anaerobic conditions. However, whether these controls are direct or indirect has been investigated only in a gene-specific manner. To gain a genomic view of anaerobic gene regulation, we determined the genome-wide in vivo DNA binding of ResD, NsrR, and Fur transcription factors (TFs) using in situ DNase I footprinting combined with chromatin affinity precipitation sequencing (ChAP-seq; genome footprinting by high-throughput sequencing [GeF-seq]). A significant number of sites were targets of ResD and NsrR, and a majority of them were also bound by Fur. The binding of multiple TFs to overlapping targets affected each individual TF's binding, which led to combinatorial transcriptional control. ResD bound to both the promoters and the coding regions of genes under its positive control. Other genes showing enrichment of ResD at only the promoter regions are targets of direct ResD-dependent repression or antirepression. The results support previous findings of ResD as an RNA polymerase (RNAP)-binding protein and indicated that ResD can associate with the transcription elongation complex. The data set allowed us to reexamine consensus sequence motifs of Fur, ResD, and NsrR and uncovered evidence that multiple TGW (where W is A or T) sequences surrounded by an A- and T-rich sequence are often found at sites where all three TFs competitively bind.IMPORTANCE Bacteria encounter oxygen fluctuation in their natural environment as well as in host organisms. Hence, understanding how bacteria respond to oxygen limitation will impact environmental and human health. ResD, NsrR, and Fur control transcription under anaerobic conditions. This work using in situ DNase I footprinting uncovered the genome-wide binding profile of the three transcription factors (TFs). Binding of the TFs is often competitive or cooperative depending on the promoters and the presence of other TFs, indicating that transcriptional regulation by multiple TFs is much more complex than we originally thought. The results from this study provide a more complete picture of anaerobic gene regulation governed by ResD, NsrR, and Fur and contribute to our further understanding of anaerobic physiology.