Cystathionine-beta-synthase inhibition for colon cancer: Enhancement of the efficacy of aminooxyacetic acid via the prodrug approach.

Colon cancer cells contain high levels of cystathionine-beta-synthase (CBS). Its product, hydrogen sulfide (H2S) promotes the growth and proliferation of colorectal tumor cells. In order to improve the antitumor efficacy of the prototypical CBS inhibitor aminooxyacetic acid (AOAA), we have designed and synthesized YD0171, a methyl ester derivative of AOAA. The antiproliferative effect of YD0171 exceeded the antiproliferative potency of AOAA in HCT116 human colon cancer cells. The esterase inhibitor paraoxon prevented the cellular inhibition of CBS activity by YD0171. YD0171 suppressed mitochondrial respiration and glycolytic function and induced G0/G1 arrest, but did not induce tumor cell apoptosis or necrosis. Metabolomic analysis in HCT116 cells showed that YD0171 affects multiple pathways of cell metabolism. The efficacy of YD0171 as an inhibitor of tumor growth was also tested in nude mice bearing subcutaneous HCT116 cancer cell xenografts. Animals were treated via subcutaneous injection of vehicle, AOAA (1, 3 or 9 mg/kg/day) or YD0171 (0.1, 0.5 or 1 mg/kg/day) for 3 weeks. Tumor growth was significantly reduced by 9 mg/kg/day AOAA, but not at the lower doses. YD0171 was more potent: tumor volume was significantly inhibited at 0.5 and 1 mg/kg/day. Thus, the in vivo efficacy of YD0171 is 9-times higher than that of AOAA. YD0171 (1 mg/kg/day) attenuated tumor growth and metastasis formation in the intracecal HCT116 tumor model. YD0171 (3 mg/kg/day) also reduced tumor growth in patient-derived tumor xenograft (PDTX) bearing athymic mice. YD0171 (3 mg/kg/day) induced the regression of established HCT116 tumors in vivo. A 5-day safety study in mice demonstrated that YD0171 at 20 mg/kg/day (given in two divided doses) does not increase plasma markers of organ injury, nor does it induce histological alterations in the liver or kidney. YD0171 caused a slight elevation in plasma homocysteine levels. In conclusion, the prodrug approach improves the pharmacological profile of AOAA; YD0171 represents a prototype for CBS inhibitory anticancer prodrugs. By targeting colorectal cancer bioenergetics, an emerging important hallmark of cancer, the approach exemplified herein may offer direct translational opportunities.

Enhanced anti-fibrogenic effects of novel oridonin derivative CYD0692 in hepatic stellate cells.

Oridonin, isolated from Rabdosia rubescens, has been proven to possess various anti-neoplastic and anti-inflammatory properties. Previously, we reported the anti-fibrogenic effects of oridonin for liver in vitro. In the present study, we investigated the effects of a newly designed analog CYD0692 in vitro. Cell viability was measured by Alamar Blue assay. Cell apoptosis was assessed by Cell Death ELISA and Yo-Pro-1 staining. Western blots were performed for cellular proteins. Flow cytometry was used to measure cell cycle regulation. CYD0692 significantly inhibited LX-2 cells proliferation in a dose- and time-dependent manner with an IC50 value of ~0.7 µM for 48 h, ~tenfold greater potency than oridonin. Similar results were observed in HSC-T6 cells. In contrast, on the human hepatocyte cell line C3A, only 12 % of the cell growth was inhibited with 5 µM of CYD0692 treatment for 48 h, while 30 % inhibited at 10 µM. After CYD0692 treatment on LX-2 cells, apoptosis and S-phase cell cycle arrest were induced; cleaved-PARP, p21, and p53 were activated while cyclin-B1 levels declined. In addition, α-smooth muscle actin, type I Collagen, and fibronectin (FN) were markedly down regulated. Transforming growth factor ß1 (TGF ß1) has been identified as a dominant stimulator for ECM production in HSC. Our results indicated that pretreatment with CYD0692 blocked TGF ß1-induced FN expression, thereby decreasing the downstream factors of TGF ß1 signaling, such as Phospho-Smad2/3 and phospho-ERK. In comparison with oridonin, its novel derivative CYD0692 has demonstrated to be a more potent and potentially safer anti-fibrogenic agent for the treatment of hepatic fibrosis.

Enhanced effects of novel oridonin analog CYD0682 for hepatic fibrosis.

BACKGROUND: Activated hepatic stellate cells (HSCs) are responsible for excess extracellular matrix (ECM) protein deposition in liver fibrosis. Previously, our group reported that the natural compound oridonin induces apoptosis, inhibits cell proliferation, and downregulates ECM proteins in activated HSC. In this study, the antifibrogenic effects of oridonin derivative CYD0682 on the activated human LX-2 and rat HSC-T6 stellate cell lines were investigated. METHODS: Cell proliferation was measured by alamarBlue assay. Apoptosis was detected by Cell Death ELISA and staining of Yo-Pro-1 and propidium iodide. Cell cycle was determined by flow cytometry. Immunoblot and immunofluorescence staining were performed for cellular protein expression. RESULTS: CYD0682 treatment significantly inhibited LX-2 cell proliferation in a dose- and time-dependent manner with an IC50 value of 0.49 µM for 48 h, ∼10-fold greater potency than oridonin. Similar results were observed in HSC-T6 cells. In contrast, 2.5 µM of CYD0682 treatment had no significant effects on proliferation of the human hepatocyte cell line C3A. CYD0682 treatment induced LX-2 cell apoptosis and S-phase cell cycle arrest and was associated with activation of p53, p21, and cleaved caspase-3. The myofibroblast marker protein α-smooth muscle actin and major ECM proteins type I collagen and fibronectin were markedly suppressed in a time- and dose-dependent fashion by CYD0682. Furthermore, pretreatment with CYD0682 blocked transforming growth factor-ß-induced type I collagen and fibronectin production. CONCLUSIONS: In comparison with oridonin, its novel derivative CYD0682 may act as a more potent antihepatic fibrosis agent.

Oridonin inhibits hepatic stellate cell proliferation and fibrogenesis.

BACKGROUND: Liver fibrosis is a common response to liver injury and, in severe cases, leads to cirrhosis. The hepatic stellate cells (HSCs) become activated after liver injury and play a significant role in fibrogenesis. The activated HSC is characterized by increased proliferation, overexpression of α smooth muscle actin, and excessive production of extracellular matrix (ECM) proteins. Oridonin, a naturally occurring diterpenoid, has been shown to induce apoptosis in liver and gastric cancer cells. However, its effects on the HSC are unknown. METHODS: We tested the effects of oridonin on the activated human and rat HSC lines LX-2 and HSC-T6, and the human hepatocyte cell line C3A. Transforming growth factor ß1 (TGF-ß1) was used to stimulate LX-2 cells. RESULTS: Oridonin significantly inhibited LX-2 and HSC-T6 proliferation. In contrast, oridonin had no antiproliferative effect on C3A cells at our tested range. Oridonin induced apoptosis and S-phase arrest in LX-2 cells. These findings were associated with an increase in p53, p21, p16, and cleaved Poly (ADP-ribose) Polymerase (PARP), and with a decrease in Cyclin-dependent kinase 4 (Cdk4). Oridonin markedly decreased expression of α smooth muscle actin and ECM protein type I collagen and fibronectin, blocked TGF-ß1-induced Smad2/3 phosphorylation and type I collagen expression. CONCLUSIONS: Oridonin induces apoptosis and cell cycle arrest involving the p53-p21 pathway in HSC and appears to be nontoxic to hepatocytes. In addition, oridonin suppressed endogenous and TGF-ß1-induced ECM proteins. Thus, oridonin may act as a novel agent to prevent hepatic fibrosis.

Heart Rate Variability Can Detect Blunt Traumatic Brain Injury Within the First Hour.

INTRODUCTION: In patients with multi-organ system trauma, the diagnosis of coinciding traumatic brain injury can be difficult due to injuries from the hemorrhagic shock that confound clinical and radiographic signs of traumatic brain injury. In this study, a novel technique using heart rate variability was developed in a porcine model to detect traumatic brain injury early in the setting of hemorrhagic shock without the need for radiographic imaging or clinical exam. METHODS: A porcine model of hemorrhagic shock was used with an arm of swine receiving hemorrhagic shock alone and hemorrhagic shock with traumatic brain injury. High-resolution heart rate frequencies were collected at different time intervals using waveforms based on voltage delivered from the heart rate monitor. Waveforms were analyzed to assess statistically significant differences between heart rate variability parameters in those with hemorrhagic shock and traumatic brain injury versus those with only hemorrhagic shock. Stochastic analysis was used to assess the validity of results and create a model by machine learning to better assess the presence of traumatic brain injury. RESULTS: Significant differences were found in several heart rate variability parameters between the two groups. Additionally, significant differences in heart rate variability parameters were found in swine within 1 hour of inducing hemorrhage in those with traumatic brain injury versus those without. These results were confirmed with stochastic analysis and machine learning was used to generate a model which determined the presence of traumatic brain injury in the setting of hemorrhage shock with 91.6% accuracy. CONCLUSIONS:  Heart rate variability represents a promising diagnostic tool to aid in the diagnosis of traumatic brain injury within 1 hour of injury.

Sepsis Increases Muscle Proteolysis in Severely Burned Adults, but Does not Impact Whole-Body Lipid or Carbohydrate Kinetics.

Sepsis is a common and often fatal consequence of severe burn injury, but its exact effects on whole body and muscle metabolism in the burn patient is unclear. To address this, 13 septic and 11 nonseptic patients (age: 36.9 ±â€Š13.0 years) with burns encompassing >30% of their total body surface area underwent muscle protein kinetic studies under postabsorptive conditions using bolus injections of ring-C6 and N phenylalanine isotopes. In parallel, whole-body lipid and carbohydrate kinetics were assessed using constant infusions of [U-C6]palmitate, [6,6-H2]glucose, and [H5]glycerol, and during a 2-h hyperinsulinemic euglycemic clamp. Muscle mRNA levels of genes implicated in the development of muscle cachexia were assessed by qPCR. Fractional breakdown rates of mixed-muscle proteins were found to be 2.4-fold greater in septic versus nonseptic patients (P < 0.05). No discernable differences in fractional synthetic rate of mixed-muscle proteins or rate of appearance of plasma free fatty acids, glycerol, or glucose could be observed between patient groups, although the latter was significantly associated with burn size (P < 0.05). Hyperinsulinemia stimulated whole-body glucose uptake and suppressed endogenous glucose production and whole-body lipolytic rate to equivalent degrees in both groups. Muscle mRNA levels of genes spanning autophagy, lysosomal, and ubiquitin proteasome-mediated proteolysis were not enhanced in septic versus nonseptic patients. Our results demonstrate that accelerated muscle proteolysis appears to be the principal metabolic consequence of sepsis in severe burn patients and could be a contributing factor to the accelerated loss of muscle mass in these individuals. The exact mechanistic basis for these changes remains unclear.

Race, Income and Insurance Status Affect Neonatal Sepsis Mortality and Healthcare Resource Utilization.

BACKGROUND: Socioeconomic disparities negatively impact neonatal health. The influence of sociodemographic disparities on neonatal sepsis is understudied. We examined the association of insurance payer status, income, race and gender on neonatal sepsis mortality and healthcare resource utilization. METHODS: We used the Kid's Inpatient Database, a nationwide population-based survey from 2006, 2009 and 2012. Neonates diagnosed with sepsis were included in the study. Multivariable logistic regression (mortality) and multivariable linear regression (length of stay and total hospital costs) were constructed to determine the association of patient and hospital characteristics. RESULTS: Our study cohort included a weighted sample of 160,677 septic neonates. Several sociodemographic disparities significantly increased mortality. Self-pay patients had increased mortality (odds ratio 3.26 [95% confidence interval: 2.60-4.08]), decreased length of stay (-2.49 ± 0.31 days, P < 0.0001) and total cost (-$5015.50 ± 783.15, P < 0.0001) compared with privately insured neonates. Additionally, low household income increased odds of death compared with the most affluent households (odds ratio 1.19 [95% confidence interval: 1.05-1.35]). Moreover, Black neonates had significantly decreased length of stay (-0.86 ± 0.25, P = 0.0005) compared with White neonates. CONCLUSIONS: This study identified specific socioeconomic disparities that increased odds of death and increased healthcare resource utilization. Moreover, this study provides specific societal targets to address to reduce neonatal sepsis mortality in the United States.

Burn Trauma Acutely Increases the Respiratory Capacity and Function of Liver Mitochondria.

BACKGROUND: A complete understanding of the role of the liver in burn-induced hypermetabolism is lacking. We investigated the acute effect of severe burn trauma on liver mitochondrial respiratory capacity and coupling control as well as the signaling events underlying these alterations. METHODS: Male BALB/c mice (8-12 weeks) received full-thickness scald burns on ∼30% of the body surface. Liver tissue was harvested 24 h postinjury. Mitochondrial respiration was determined by high-resolution respirometry. Citrate synthase activity was determined as a proxy of mitochondrial density. Male Sprague-Dawley rats received full-thickness scald burns to ∼60% of the body surface. Serum was collected 24 h postinjury. HepG2 cells were cultured with serum-enriched media from either sham- or burn-treated rats. Protein levels were analyzed via western blot. RESULTS: Mass-specific (P = 0.01) and mitochondrial-specific (P = 0.01) respiration coupled to ATP production significantly increased in the liver after burn. The respiratory control ratio for ADP (P = 0.04) and the mitochondrial flux control ratio (P = 0.03) were elevated in the liver of burned animals. Complex III and Complex IV protein abundance in the liver increased after burn by 17% and 14%, respectively. Exposure of HepG2 cells to serum from burned rats increased the pAMPKα:AMPKα ratio (P < 0.001) and levels of SIRT1 (P = 0.01), Nrf2 (P < 0.001), and PGC1α (P = 0.02). CONCLUSIONS: Severe burn trauma augments respiratory capacity and function of liver mitochondria, adaptations that augment ATP production. This response may be mediated by systemic factors that activate signaling proteins responsible for regulating cellular energy metabolism and mitochondrial biogenesis.

Health Disparities in Adolescent Bariatric Surgery: Nationwide Outcomes and Utilization.

PURPOSE: Bariatric surgery represents an appropriate treatment for adolescent severe obesity, but its utilization remains low in this patient population. We studied the impact of race and sex on preoperative characteristics, outcomes, and utilization of adolescent bariatric surgery. METHODS: Retrospective analysis (2007-2014) of adolescent bariatric surgery using the Bariatric Outcomes Longitudinal Database, a national database that collects bariatric surgical care data. We assessed the relationships between baseline characteristics and outcomes (weight loss and remission of obesity-related conditions [ORCs]). Using the National Health and Nutrition Examination Survey and U.S. census data, we calculated the ratio of severe obesity and bariatric procedures among races and determined the ratio of ratios to assess for disparities. RESULTS: About 1,539 adolescents underwent bariatric surgery. Males had higher preoperative body mass index (BMI; 51.8 ± 10.5 vs. 47.1 ± 8.7, p < .001) and higher rates of obstructive sleep apnea and dyslipidemia. Blacks had higher preoperative BMI (52.4 ± 10.6 vs. 47.3 ± 8.3; 48.7 ± 8.8; 48.2 ± 12.1 kg/m2; whites, Hispanics, and others, respectively p < .001) and higher rates of hypertension, obstructive sleep apnea, and asthma. Weight loss and ORCs remission rates did not differ between sexes or races after accounting for the rate of severe obesity in each racial group. White adolescents underwent bariatric surgery at a higher proportion than blacks and Hispanics (2.5 and 2.3 times higher, respectively). CONCLUSIONS: Preoperative characteristics vary according to race and sex. Race and sex do not impact 12-month weight loss or ORC's remission rates. Minority adolescents undergo bariatric surgery at lower-than-expected rates.

Race and outcomes in gastroschisis repair: a nationwide analysis.

BACKGROUND: The incidence of gastroschisis has increased 30% between the periods 1995-2005 and 2006-2012, with the largest increase in Black neonates born to Black mothers younger than 20years old. OBJECTIVE: Racial disparities in peri- and post-operative outcomes have been previously identified in several types of adult and pediatric surgical patients. Is there an association between race and clinical outcomes and healthcare resource utilization in neonates with gastroschisis? METHODS: Retrospective study using national administrative data from the Kid's Inpatient Database (KID) from 2006, 2009, and 2012 for neonates (age<28days) with gastroschisis. Multivariable logistic regression was constructed to determine the association of race and socioeconomic characteristics with complications and mortality; linear regression was used for length of stay and hospital charges. RESULTS: We identified 3846 neonates with gastroschisis that underwent surgical repair, including 676 patients with complex gastroschisis. When controlling for birth weight, payer status, socioeconomic status, and hospital characteristics, Black neonates had increased odds of having complex gastroschisis and associated atresias. Mortality was higher in patients with complex gastroschisis, patients from the lowest income quartiles, and patients with Medicaid as primary payer (compared to those with private insurance). Length of stay (LOS) was increased in patients with complex gastroschisis, birth weight <2500g, and Medicaid patients. Hospital charges were higher in complex gastroschisis, Black and Hispanic neonates (as compared to Whites), males, birth weight <2500g, and Medicaid patients. CONCLUSIONS: There is an association between race and complex gastroschisis, associated intestinal atresias, and total charges in neonates with gastroschisis. In addition, income status is associated with mortality and hospital charges while payer status is associated with complications, mortality, LOS, and hospital charges. Public health and prenatal interventions should target at-risk populations to improve clinical outcomes. PROGNOSIS STUDY: Level of Evidence: II.

STAT3 Inhibition Suppresses Hepatic Stellate Cell Fibrogenesis: HJC0123, a Potential Therapeutic Agent for Liver Fibrosis.

Hepatic Stellate Cells (HSCs) are the major source of the excessive extracellular matrix (ECM) production that replaces liver parenchyma with fibrous tissue during liver fibrosis. The signal transducer and activator of transcription 3 (STAT3) promotes HCSs survival, proliferation, and activation contributing to fibrogenesis. We have previously used a fragment-based drug design approach and have discovered a novel STAT3 inhibitor, HJC0123. Here, we explored the biological effects of HJC0123 on the fibrogenic properties of HSCs. HJC0123 treatment resulted in the inhibition of HSCs proliferation at submicromolar concentrations. HJC0123 reduced the phosphorylation, nuclear translocation, and transcriptional activity of STAT3. It decreased the expression of STAT3-regulated proteins, induced cell cycle arrest, promoted apoptosis and downregulated SOCS3. HJC0123 treatment inhibited HSCs activation and downregulated ECM protein fibronectin and type I collagen expression. In addition, HJC0123 increased IL-6 production and decreased TGF-ß induced Smad2/3 phosphorylation. These results demonstrate that HJC0123 represents a novel STAT3 inhibitor that suppresses the fibrogenic properties of HSCs, suggesting its therapeutic potential in liver fibrosis.

Transpulmonary Thermodilution Versus Transthoracic Echocardiography for Cardiac Output Measurements in Severely Burned Children.

INTRODUCTION: Severe burns trigger a hyperdynamic state, necessitating accurate measurement of cardiac output (CO) for cardiovascular observation and guiding fluid resuscitation. However, it is unknown whether, in burned children, the increasingly popular transthoracic echocardiography (TTE) method of CO measurement is as accurate as the widely used transpulmonary thermodilution (TPTD) method. PATIENTS AND METHODS: We retrospectively compared near-simultaneously performed CO measurements in severely burned children using TPTD with the Pulse index Continuous Cardiac Output (PiCCO) system or TTE. Outcomes were compared using t tests, multiple linear regression, and a Bland-Altman plot. RESULTS: Fifty-four children (9 ±â€Š5 years) with 68 ±â€Š18% total body surface area burns were studied. An analysis of 105 data pairs revealed that PiCCO yielded higher CO measurements than TTE (190 ±â€Š39% vs. 150 ±â€Š50% predicted values; P < 0.01). PiCCO- and TTE-derived CO measurements correlated moderately well (R = 0.54, P < 0.01). A Bland-Altman plot showed a mean bias of 1.53 L/min with a 95% prediction interval of 4.31 L/min. CONCLUSIONS: TTE-derived estimates of CO may underestimate severity of the hyperdynamic state in severely burned children. We propose using the PiCCO system for objective cardiovascular monitoring and to guide goal-directed fluid resuscitation in this population.

BurnCase 3D software validation study: Burn size measurement accuracy and inter-rater reliability.

OBJECTIVE: The aim of this study was to compare the accuracy of burn size estimation using the computer-assisted software BurnCase 3D (RISC Software GmbH, Hagenberg, Austria) with that using a 2D scan, considered to be the actual burn size. METHODS: Thirty artificial burn areas were pre planned and prepared on three mannequins (one child, one female, and one male). Five trained physicians (raters) were asked to assess the size of all wound areas using BurnCase 3D software. The results were then compared with the real wound areas, as determined by 2D planimetry imaging. To examine inter-rater reliability, we performed an intraclass correlation analysis with a 95% confidence interval. RESULTS: The mean wound area estimations of the five raters using BurnCase 3D were in total 20.7±0.9% for the child, 27.2±1.5% for the female and 16.5±0.1% for the male mannequin. Our analysis showed relative overestimations of 0.4%, 2.8% and 1.5% for the child, female and male mannequins respectively, compared to the 2D scan. The intraclass correlation between the single raters for mean percentage of the artificial burn areas was 98.6%. There was also a high intraclass correlation between the single raters and the 2D Scan visible. CONCLUSION: BurnCase 3D is a valid and reliable tool for the determination of total body surface area burned in standard models. Further clinical studies including different pediatric and overweight adult mannequins are warranted.

Skeletal Muscle Protein Breakdown Remains Elevated in Pediatric Burn Survivors up to One-Year Post-Injury.

Acute alterations in skeletal muscle protein metabolism are a well-established event associated with the stress response to burns. Nevertheless, the long-lasting effects of burn injury on skeletal muscle protein turnover are incompletely understood. This study was undertaken to investigate fractional synthesis (FSR) and breakdown (FBR) rates of protein in skeletal muscle of pediatric burn patients (n  =  42, >30% total body surface area burns) for up to 1 year after injury. Skeletal muscle protein kinetics were measured in the post-prandial state following bolus injections of C6 and N phenylalanine stable isotopes. Plasma and muscle phenylalanine enrichments were quantified using gas chromatography-mass spectrometry. We found that the FSR in burn patients was 2- to 3-fold higher than values from healthy men previously reported in the literature (P ≤ 0.05). The FBR was 4- to 6-fold higher than healthy values (P  <  0.01). Therefore, net protein balance was lower in burn patients compared with healthy men from 2 weeks to 12 months post-injury (P  <  0.05). These findings show that skeletal muscle protein turnover stays elevated for up to 1 year after burn, an effect attributable to simultaneous increases in FBR and FSR. Muscle FBR exceeds FSR during this time, producing a persistent negative net protein balance, even in the post-prandial state, which likely contributes to the prolonged cachexia seen in burned victims.

Heart rate variability analysis is more sensitive at identifying neonatal sepsis than conventional vital signs.

BACKGROUND: Sepsis remains the largest preventable source of neonatal mortality in the world. Heart rate variability (HRV) analysis and noninvasive cardiac output have been shown to be useful adjuncts to sepsis detection in many patient groups. METHODS: With Institutional Review Board approval, 4 septic and 6 nonseptic extremely low birth weight patients were enrolled. Data from septic and healthy patients were collected for 5 hours. Electrocardiogram waveform and traditional vital signs were collected and the RR intervals were calculated; then HRV analysis was performed in both the time and frequency domain. RESULTS: HRV measurements in time domain, heart rate, and pulse oximetry (SpO2) were significantly different in septic patients vs nonseptic controls. CONCLUSIONS: These results indicate that nonconventional vital signs such as HRV are more sensitive than traditionally used vital signs, such as cardiac output and mean arterial pressure, in the confirmation of sepsis in extremely low birth weight neonates. HRV may allow for earlier identification of septic physiology.

Biochemical and genetic characterization of an early step in a novel pathway for the biosynthesis of aromatic amino acids and p-aminobenzoic acid in the archaeon Methanococcus maripaludis.

Methanococcus maripaludis is a strictly anaerobic, methane-producing archaeon and facultative autotroph capable of biosynthesizing all the amino acids and vitamins required for growth. In this work, the novel 6-deoxy-5-ketofructose-1-phosphate (DKFP) pathway for the biosynthesis of aromatic amino acids (AroAAs) and p-aminobenzoic acid (PABA) was demonstrated in M. maripaludis. Moreover, PABA was shown to be derived from an early intermediate in AroAA biosynthesis and not from chorismate. Following metabolic labelling with [U-(13)C]-acetate, the expected enrichments for phenylalanine and arylamine derived from PABA were observed. DKFP pathway activity was reduced following growth with aryl acids, an alternative source of the AroAAs. Lastly, a deletion mutant of aroA', which encodes the first step in the DKFP pathway, required AroAAs and PABA for growth. Complementation of the mutants by an aroA' expression vector restored the wild-type phenotype. In contrast, a deletion of aroB', which encodes the second step in the DKFP pathway, did not require AroAAs or PABA for growth. Presumably, methanococci contain an alternative activity for this step. These results identify the initial reactions of a new pathway for the biosynthesis of PABA in methanococci.