Validation of the Artificial Intelligence Prognostic Scoring System for Myelodysplastic Syndromes in chronic myelomonocytic leukaemia: A novel approach for improved risk stratification.

Chronic myelomonocytic leukaemia (CMML) is a rare haematological disorder characterized by monocytosis and dysplastic changes in myeloid cell lineages. Accurate risk stratification is essential for guiding treatment decisions and assessing prognosis. This study aimed to validate the Artificial Intelligence Prognostic Scoring System for Myelodysplastic Syndromes (AIPSS-MDS) in CMML and to assess its performance compared with traditional scores using data from a Spanish registry (n = 1343) and a Taiwanese hospital (n = 75). In the Spanish cohort, the AIPSS-MDS accurately predicted overall survival (OS) and leukaemia-free survival (LFS), outperforming the Revised-IPSS score. Similarly, in the Taiwanese cohort, the AIPSS-MDS demonstrated accurate predictions for OS and LFS, showing superiority over the IPSS score and performing better than the CPSS and molecular CPSS scores in differentiating patient outcomes. The consistent performance of the AIPSS-MDS across both cohorts highlights its generalizability. Its adoption as a valuable tool for personalized treatment decision-making in CMML enables clinicians to identify high-risk patients who may benefit from different therapeutic interventions. Future studies should explore the integration of genetic information into the AIPSS-MDS to further refine risk stratification in CMML and improve patient outcomes.

Baseline Severity and Inflammation Would Influence the Effect of Simvastatin on Clinical Outcomes in Cirrhosis Patients.

BACKGROUND: Simvastatin administration to decompensated cirrhosis patients improved Child-Pugh (CP) at the end of a safety trial (EST). AIM: To evaluate whether simvastatin reduces cirrhosis severity through a secondary analysis of the safety trial. METHODS: Thirty patients CP class (CPc) CPc A (n = 6), CPc B (n = 22), and CPc C (n = 2) received simvastatin for one year. PRIMARY ENDPOINT: cirrhosis severity. Secondary endpoints: health-related quality of life (HRQoL) and hospitalizations for cirrhosis complications. RESULTS: Cirrhosis severity decreased baseline versus EST only across CP score (7.3 ± 1.3 versus 6.7 ± 1.7, P = 0.041), and CPc: 12 patients lessened from CPc B to CPc A, and three patients increased from CPc A to CPc B (P = 0.029). Due to cirrhosis severity changes and differences in clinical outcomes, 15 patients completed the trial as CPc AEST and another 15 as CPc B/C. At baseline, CPc AEST showed greater albumin and high-density lipoprotein cholesterol concentrations than CPc B/C (P = 0.036 and P = 0.028, respectively). Comparing EST versus baseline, only in CPc AEST, there was a reduction in white-cell blood (P = 0.012), neutrophils (P = 0.029), monocytes (P = 0.035), and C-reactive protein (P = 0.046); an increase in albumin (P = 0.011); and a recovery in HRQoL (P < 0.030). Finally, admissions for cirrhosis complications decreased in CPc AEST versus CPc B/C (P = 0.017). CONCLUSIONS: Simvastatin would reduce cirrhosis severity only in CPc B at baseline in a suitable protein and lipid milieu, possibly due to its anti-inflammatory effects. Furthermore, only in CPc AEST would improve HRQoL and reduce admissions by cirrhosis complications. However, as these outcomes were not primary endpoints, they require validation.

Safety of Chronic Simvastatin Treatment in Patients with Decompensated Cirrhosis: Many Adverse Events but No Liver Injury.

BACKGROUND: The high mortality rate of decompensated cirrhosis underlines the need for new treatments. Experimental models of cirrhosis and its reported relationship with atherosclerotic cardiovascular disease have provided data supporting the rational use of statins in these patients. However, little is known about the safety of statins in this setting. AIM: We evaluate the safety of chronic simvastatin treatment in patients with decompensated cirrhosis. METHODS: We conducted a prospective, open, uncontrolled, phase 2a trial in 30 patients with Child-Pugh class A (n = 6), B (n = 22), and C (n = 2) decompensated cirrhosis. The patients received standard treatment throughout the trial plus simvastatin 20 mg/day for 2 weeks and thereafter simvastatin 40 mg/day up to 1 year. RESULTS: Sixteen out of 30 patients (53.3%) showed adverse events, including gastrointestinal toxicity (36.7%), muscle injury (MI) (36.7%), and headache (13.3%). No liver injury was registered. Due to MI alone, simvastatin dosage was reduced in 23.4% of cases and transiently interrupted in 13.3%. Once these adverse events were overcome, simvastatin was resumed until the end of the trial. MI was associated with baseline MELD score > 12 (p = 0.035) and with baseline Child-Pugh class C. No MI was associated with final Child-Pugh score ≤ 6 (p = 0.030) or final Child-Pugh class A (p = 0.020). CONCLUSIONS: Chronic treatment with simvastatin 40 mg/day in patients with decompensated cirrhosis was associated with several adverse events, being MI the only clinically significant one, which appears to be related to the simvastatin dosage and the degree of cirrhosis severity. Noticeably, no liver injury was recorded.

Surveillance for Hepatocellular Carcinoma: Does the Place Where Ultrasound Is Performed Impact Its Effectiveness?

BACKGROUND: Biannual ultrasound (US) is recommended as the clinical screening tool for hepatocellular carcinoma (HCC). The effectiveness of surveillance according to the place where US is performed has not been previously reported. AIMS: To compare the effectiveness of US performed in the center responsible for follow-up as opposed to US proceeding from centers other than that of follow-up. METHODS: This is a multicenter cohort study from Argentina. The last US was categorized as done in the same center or done in a different center from the institution of the patient's follow-up. Surveillance failure was defined as HCC diagnosis not meeting Barcelona Clinic Liver Cancer (BCLC) stages 0-A or when no nodules were observed at HCC diagnosis. RESULTS: From 533 patients with HCC, 62.4% were under routine surveillance with a surveillance failure of 38.8%. After adjusting for a propensity score matching, BCLC stage and lead-time survival bias, surveillance was associated with a significant survival benefit [HR of 0.51 (CI 0.38; 0.69)]. Among patients under routine surveillance (n = 345), last US was performed in the same center in 51.6% and in a different center in 48.4%. Similar rates of surveillance failure were observed between US done in the same or in a different center (32% vs. 26.3%; P = 0.25). Survival was not significantly different between both surveillance modalities [HR 0.79 (CI 0.53; 1.20)]. CONCLUSIONS: Routine surveillance for HCC in the daily practice improved survival either when performed in the same center or in a different center from that of patient's follow-up.

Refining risk prediction in pediatric acute lymphoblastic leukemia through DNA methylation profiling.

Acute lymphoblastic leukemia (ALL) is the most prevalent cancer in children, and despite considerable progress in treatment outcomes, relapses still pose significant risks of mortality and long-term complications. To address this challenge, we employed a supervised machine learning technique, specifically random survival forests, to predict the risk of relapse and mortality using array-based DNA methylation data from a cohort of 763 pediatric ALL patients treated in Nordic countries. The relapse risk predictor (RRP) was constructed based on 16 CpG sites, demonstrating c-indexes of 0.667 and 0.677 in the training and test sets, respectively. The mortality risk predictor (MRP), comprising 53 CpG sites, exhibited c-indexes of 0.751 and 0.754 in the training and test sets, respectively. To validate the prognostic value of the predictors, we further analyzed two independent cohorts of Canadian (n = 42) and Nordic (n = 384) ALL patients. The external validation confirmed our findings, with the RRP achieving a c-index of 0.667 in the Canadian cohort, and the RRP and MRP achieving c-indexes of 0.529 and 0.621, respectively, in an independent Nordic cohort. The precision of the RRP and MRP models improved when incorporating traditional risk group data, underscoring the potential for synergistic integration of clinical prognostic factors. The MRP model also enabled the definition of a risk group with high rates of relapse and mortality. Our results demonstrate the potential of DNA methylation as a prognostic factor and a tool to refine risk stratification in pediatric ALL. This may lead to personalized treatment strategies based on epigenetic profiling.

TECIPOT: Study for the Evaluation of New Technologies and Process Engineering Applied to the Optimization of Hospital Transfers.

There is a need for coordinating and manage the information flow between clinical, nursing, ancillary, cleaning and administrative staff to optimize patient transfers during admission and discharge. TECIPOT project proposes the incorporation of new technologies associated with the optimization of emergency circuits to improve the efficiency in the management of patients in the center, as well as improve the perception of patients and professionals about the processes of transfers of emergency patients, observation and discharge from the Hospital. The project proposes a 24 month study to evaluate the impact on healthcare processes.

Clinical Outcomes after Surgical Resection Combined with Brachytherapy for Uveal Melanomas.

Currently, brachytherapy is the most commonly used therapeutic approach for uveal melanomas. Surgical resection by means of endoresection or exoresection is an alternative approach. The present report recounts our experience over 15 years in the treatment of uveal melanoma using a combined approach of resection surgery with brachytherapy. This is a single-center observational retrospective cohort study in which we describe clinical outcomes, complications and survival in 35 cases of melanoma of the iris or the ciliary body after a combination of surgery and brachytherapy or brachytherapy alone. Local treatment of the tumor was successful in all cases with surgery and brachytherapy. The most frequent complications were scleromalacia, bullous keratopathy, retinal toxicity, cataracts, hypotonia, and photophobia. There were three cases of recurrence, all of which were found in the group of patients who had received brachytherapy alone, and in one case we had to perform a secondary enucleation due to tumor growth after brachytherapy. At present, only one patient has died during follow-up due to liver metastases six years after the start of treatment. In carefully selected patients, this approach can be effective and safe, as long as a close follow-up is carried out after surgery.

Novel, versatile, and tightly regulated expression system for Escherichia coli strains.

A novel tightly regulated gene expression system was developed for Escherichia coli by applying the regulatory elements of the Pseudomonas putida F1 cym and cmt operons to control target gene expression at the transcriptional level by using p-isopropylbenzoate (cumate) as an inducer. This novel expression system, referred to as the cumate gene switch, includes a specific expression vector, pNEW, that contains a partial T5 phage promoter combined with the Pseudomonas-based synthetic operator and the cymR repressor protein-encoding gene designed to express constitutively in the host strain. The induction of transcription relies on the addition of the exogenous inducer (cumate), which is nontoxic to the culture, water soluble, and inexpensive. The characteristics and potential of the expression system were determined. Using flow cytometry and fed-batch fermentations, we have shown that, with the newly developed cumate-regulated system, (i) higher recombinant product yields can be obtained than with the pET (isopropyl-beta-D-thiogalactopyranoside [IPTG])-induced expression system, (ii) expression is tightly regulated, (iii) addition of cumate quickly results in a fully induced and homogenous protein-expressing population in contrast to the bimodal expression profile of an IPTG-induced population, (iv) expression can be modulated by varying the cumate concentration, and (v) the cumate-induced population remains induced and fully expressing even at 8 h following induction, resulting in high yields of the target protein Furthermore, the cumate gene switch described in this article is applicable to a wide range of E. coli strains.

Production of functionalized polyhydroxyalkanoates by genetically modified Methylobacterium extorquens strains.

BACKGROUND: Methylotrophic (methanol-utilizing) bacteria offer great potential as cell factories in the production of numerous products from biomass-derived methanol. Bio-methanol is essentially a non-food substrate, an advantage over sugar-utilizing cell factories. Low-value products as well as fine chemicals and advanced materials are envisageable from methanol. For example, several methylotrophic bacteria, including Methylobacterium extorquens, can produce large quantities of the biodegradable polyester polyhydroxybutyric acid (PHB), the best known polyhydroxyalkanoate (PHA). With the purpose of producing second-generation PHAs with increased value, we have explored the feasibility of using M. extorquens for producing functionalized PHAs containing C-C double bonds, thus, making them amenable to future chemical/biochemical modifications for high value applications. RESULTS: Our proprietary M. extorquens ATCC 55366 was found unable to yield functionalized PHAs when fed methanol and selected unsaturated carboxylic acids as secondary substrates. However, cloning of either the phaC1 or the phaC2 gene from P. fluorescens GK13, using an inducible and regulated expression system based on cumate as inducer (the cumate switch), yielded recombinant M. extorquens strains capable of incorporating modest quantities of C-C double bonds into PHA, starting from either C6= and/or C8=. The two recombinant strains gave poor results with C11=. The strain containing the phaC2 gene was better at using C8= and at incorporating C-C double bonds into PHA. Solvent fractioning indicated that the produced polymers were PHA blends that consequently originated from independent actions of the native and the recombinant PHA synthases. CONCLUSIONS: This work constitutes an example of metabolic engineering applied to the construction of a methanol-utilizing bacterium capable of producing functionalized PHAs containing C-C double bonds. In this regard, the PhaC2 synthase appeared superior to the PhaC1 synthase at utilizing C8= as source of C-C double bonds and at incorporating C-C double bonds into PHA from either C6= or C8=. The M. ex-phaC2 strain is, therefore, a promising biocatalyst for generating advanced (functionalized) PHAs for future high value applications in various fields.

Etiology of hepatocellular carcinoma in Latin America: a prospective, multicenter, international study.

BACKGROUND/AIMS: No prospective study has been published investigating etiology of HCC in Latin America. The primary aim of this prospective study was to analyze the etiology of liver disease in patients with HCC from our area. Secondary aims were to evaluate staging using Okuda and BCLC classifications; and percentage of patients receiving treatment. METHODS: The Governing Board of the Latin American Association for the Study of the Liver designed the protocol. During a 18 month period, all members were invited to load their incident HCC cases on line. RESULTS: 240 cases from 9 countries were uploaded, 174 were male (72.5%), median age was 64 years, interquartile range 57-72. In 85.4% of cases, patients had underlying cirrhosis. Main etiological factors were: HCV in 74 patients (30.8%), alcohol in 49 (20.4%), cryptogenic cirrhosis in 35 (14.6%), HBV in 26 (10.8%), HCV plus alcohol in 14 (5.8%). Considering the combinations, hepatitis C was shown in 91 patients (38%); chronic alcoholism in 68 patients (28%); and hepatitis B in 33 patients (14%). There were no significant differences between the groups in the age at diagnosis. Percentage of male gender was higher in groups of alcohol (94%), HCV plus alcohol (93%) and HBV (85%) than in cryptogenic cirrhosis (60%) and HCV (59%) (p<0.001). CONCLUSIONS: Our prospective study showed that hepatitis C is the more frequent etiology of HCC in Latin America, followed by alcoholic cirrhosis. Demographical results showed a male predominance (male:female ratio 2.6) with an important proportion of patients being diagnosed at their sixties.

Serum creatinine and bilirubin predict renal failure and mortality in patients with spontaneous bacterial peritonitis: a retrospective study.

BACKGROUND: Patients with spontaneous bacterial peritonitis (SBP) are at a high risk for renal failure and death despite successful treatment of infection. Intravenous (IV) albumin administration combined with antibiotic treatment has been shown to significantly decrease these risks. Clinical evidence is lacking on which patients are appropriate candidates for albumin treatment. AIM: To retrospectively analyse the usefulness of serum creatinine and bilirubin levels in predicting renal failure and mortality of patients hospitalized for SBP. METHODS: Between March 1995 and September 1998, 127 cirrhotic patients with SBP who had not received plasma expansion were evaluated. Eighty-one patients (64%) were classified as having a high risk for renal failure and mortality (serum bilirubin >4 mg/dl or serum creatinine >1 mg/dl) and 46 (36%) as having a low risk. RESULTS: At admission, 36.3% of all patients presented renal failure. Mortality during their hospitalization was 23% among those with a high risk and 6.5% among those with a low risk (P=0.01). Renal failure occurred in 23% of the high-risk patients, compared with 2.6% of the low-risk patients (P=0.006). The presence of hyponatraemia was significantly associated with higher mortality and renal failure in the high-risk group. CONCLUSIONS: Our retrospective review of patients with SBP suggests that serum bilirubin levels >4 mg and serum creatinine levels >1 mg/dl at the time of diagnosis represent significant risk factors for the clinical outcomes of patients with SBP. Patients without these risk factors may have a very low likelihood of death or renal failure.

Phylogenetic analysis of methanotrophic communities in cover soils of a landfill in Ontario.

We examined the methanotrophs in the Trail Road Landfill soils, Ottawa, Ontario, through cultivation-independent molecular assay and the culturing approach. Denaturing gradient gel electrophoresis (DGGE) analysis of amplified methanotroph-specific 16S rDNA gene fragments revealed a more diverse type I (RuMP pathway) methanotrophic community than type II (serine pathway) in 17 soil samples taken along a 50 m transect. The type II methanotrophic community was less diverse, with the dominance of Methylocystis in almost all samples, and clustering with high similarity (85%-88%). Also, the results showed that the C/N ratio of soil organic matter could significantly affect the methanotrophic community structures. The DGGE results were supported by sequence analysis of cloned pmoA. Members of the genera Methylobacter (type I), Methylocaldum (type X), and Methylocystis (type II) appeared to be the dominant methanotrophs. From methanotrophic enrichments, we isolated type I Methylobacter sp., and 3 type II Methylocystis spp.,which appeared to be one of the dominant bacteria species in the soil sample from which isolates were obtained.

Etiology of hepatocellular carcinoma in Argentina: results of a multicenter retrospective study.

Incidence and etiology of hepatocellular carcinoma (HCC) are variable around the world, depending mainly on theprevalence ofchronic hepatitis B carriers in each region. No study has been published analyzing epidemiological features of patients with HCC in Argentina. The aim of this retrospective study was to describe demographical and etiological results in a series of 587 consecutive patients with HCC diagnosed in 15 Hepatology and Gastroenterology Units distributed all around our country. Seventy-two per cent of patients were male, the median age was 62 years (interquartile range 55-68 years), and 93% had cirrhosis. Regarding to etiological data (fully available in 551 cases), main etiologies were chronic alcoholism in 229 patients (41.6%) (the sole risk factor in 182, associated to HCVin 35 and to HBV in 12); hepatitis C in 223 patients (40.5%) (the sole risk factor in 181, associated to alcoholism in 35 and to HBV in 7); hepatitis B in 74 patients (13.4%) (the sole risk factor in 55, associated to alcoholism in 12 and to HCV in 7); cryptogenic cirrhosis in 51 patients (9.2%). There were significant differences in percentages of genders between main groups: males were highly predominant in alcoholic cirrhosis (93%), hepatitis B (87%) and HCV plus alcohol (94%), compared to 63% in cryp togenic cirrhosis and 49% in hepatitis C (p<0.01). There were no differences in age at presentation between the main etiologies. In conclusion, the main causes of HCC in Argentina are alcoholic cirrhosis and hepatitis C (76% of cases). A majority of patients with HCC in our country are cirrhotics, males, and in their 6th or -7th decades of life.

Fluorescence-based real time monitoring and diagnostics of recombinant Pichia pastoris cultivations in a bioreactor.

This study describes the application of the multivariate curve resolution (MCR) analysis technique for real-time analysis of culture fluorescence during recombinant Pichia pastoris cultivation in a bioreactor. Fluorescence spectra were acquired with an on-line dual excitation wavelength fluorometer and then used to develop a real time MCR-based bioprocess monitoring and diagnostics tool. Initial bioreactor experiments using two similar recombinant antibody secreting P. pastoris cell lines showed significant differences in protein production. To distinguish between the contributions of operating conditions and the specific cell line's genetic composition to the observed differences in protein production, the bioreactor experiments were repeated and accompanied by real time MCR analysis. The tests demonstrated high sensitivity of MCR-derived "pure concentration" profiles to growth as well as to initial conditions, thus enabling real-time cultivation process trend diagnostics and fault detection. © 2018 Her Majesty the Queen in Right of Canada © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2761, 2019.

Production of an insecticidal crystal protein from Bacillus thuringiensis by the methylotroph Methylobacterium extorquens.

The Cry1Aa protein from Bacillus thuringiensis is an insecticidal protein that is highly active against several species of Lepidoptera. We cloned and expressed the cry1Aa gene in a plant-colonizing methylotroph, Methylobacterium extorquens, under the control of the strong M. extorquens AM1 methanol dehydrogenase promoter, P(mxaF). Transmission electron microscopy revealed characteristic bipyramidal intracellular delta-endotoxin crystals similar to the crystalline inclusions formed by B. thuringiensis. Both the protoxin protein and the activated toxin were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis. In single-dose assays of the recombinant against the silkworm, Bombyx mori, both whole cells and cell lysates caused rapid feeding inhibition followed by mortality. The biomass and growth rate of recombinant cells in shake flask culture were similar to those of the wild-type strain, indicating a lack of fitness cost to the recombinant under controlled culture conditions. Recombinant Cry1Aa was expressed at a level of 4.5% of total M. extorquens cell protein. The potential benefits of modifying M. extorquens to deliver insecticidal Cry proteins for crop and forest protection are discussed.

Prevalence of primary sclerosing cholangitis in patients with ulcerative colitis and the risk of developing malignancies. A large prospective study.

BACKGROUND/AIM: primary sclerosing cholangitis (PSC) is associated with ulcerative colitis (UC) and seems to be a risk factor for colon cancer. However, taking into account that no data are available in South American population, we analyzed the prevalence of PSC in 1,333 patients with UC and the risk for developing colon cancer. MATERIAL: patients with persistent increases of alkaline phosphatase were studied by cholangiography and liver biopsy. To assess the risk of colon cancer, each patient with PSC and UC was matched with two control patients with UC without PSC of the same age, gender, extent and duration of UC. RESULTS: the whole prevalence of PSC was 2.9% (39 patients) reaching 6.2% in extensive colitis. Seven (18%) out of 39 patients with PSC developed colorectal carcinoma compared with 2 out of 78 (2.6%) in the control group (p=0.006). The cumulative risk of colorectal carcinoma was 11% and 18% after 10 and 20 years in the PSC group compared with 2% and 7% in the control group, respectively (p=0.002). CONCLUSION: this is the first prospective study performed in Latin America showing that the prevalence of PSC in patients with UC is similar to that reported in the Anglo-Saxon population. Patients with UC and PSC have a high risk of colorectal cancer.

Adherence to Barcelona Clinic Liver Cancer therapeutic algorithm for hepatocellular carcinoma in the daily practice: a multicenter cohort study from Argentina.

BACKGROUND AND AIM: Adherence to the Barcelona Clinic Liver Cancer (BCLC) staging algorithm for the treatment of hepatocellular carcinoma is challenging in the daily practice. We aimed to analyze adherence to BCLC along with its effect on patient survival. PATIENTS AND METHODS: A cohort study was conducted in 14 hospitals from Argentina including patients with newly diagnosed hepatocellular carcinoma (2009-2016). Adherence was considered when the first treatment was the one recommended by the BCLC. RESULTS: Overall, 708 patients were included. At diagnosis, BCLC stages were as follows: stage 0 4%, A 43%, B 22%, C 9% and D 22%. Overall, 53% of the patients were treated according to BCLC, 24% were undertreated, and 23% overtreated. Adherence to BCLC increased to 63% in subsequent treatments. Independent factors associated with adherence to BCLC were the presence of portal hypertension [odds ratio: 1.63; 95% confidence interval (CI): 1.11-2.39] and BCLC stage C (odds ratio: 0.32; 95% CI: 0.12-0.72). In a multivariable model adjusting for portal hypertension and BCLC stages, adherence to BCLC showed improved survival (hazard ratio: 0.67; 95% CI: 0.52-0.87). CONCLUSION: Adherence to BCLC represents a challenge in the daily practice, with almost half of the patients being treated accordingly, showing that the decision-making process should be tailored to each individual patient.

Clinical implementation of combined modulated electron and photon beams with conventional MLC for accelerated partial breast irradiation.

PURPOSE: To report the clinical implementation of a novel external beam radiotherapy technique for accelerated partial breast irradiation treatments based on combined electron and photon modulated beams radiotherapy (MERT+IMRT) with conventional MLC. MATERIALS AND METHODS: A group of patients was selected to test the viability of the technique. The prescribed dose was 38.5Gy, following a hypofractionated schema, and the structures were defined following the NSABP-B39/RTOG-0413 protocol. The plans were calculated with an in-house Monte Carlo based planning system to consider explicitly the particle interactions with the MLC. An ad-hoc breast phantom was designed for a specific QA protocol. A reduced SSD was used for electron beams. Toxicity and cosmetic effects were assessed at every follow-up visit. RESULTS: All the plans achieved the dosimetric objectives and fulfilled the specific quality assurance protocol. Treatment delivery did not entail additional drawbacks for the clinical routine. Moderate or severe grade of toxicity was not reported, and the cosmetic results were comparable to those obtained with other APBI techniques. CONCLUSIONS: Results showed that MERT+IMRT with the MLC is a feasible and secure technique, and easy to be extended to other centers with the implementation of the adequate software for planning.

Diversity of soluble methane monooxygenase-containing methanotrophs isolated from polluted environments.

Methanotrophs were enriched and isolated from polluted environments in Canada and Germany. Enrichments in low copper media were designed to specifically encourage growth of soluble methane monooxygenase (sMMO) containing organisms. The 10 isolates were characterized physiologically and genetically with one type I and nine type II methanotrophs being identified. Three key genes: 16S rRNA; pmoA and mmoX, encoding for the particulate and soluble methane monooxygenases respectively, were cloned from the isolates and sequenced. Phylogenetic analysis of these sequences identified strains, which were closely related to Methylococcus capsulatus, Methylocystis sp., Methylosinus sporium and Methylosinus trichosporium. Diversity of sMMO-containing methanotrophs detected in this and previous studies was rather narrow, both genetically and physiologically, suggesting possible constraints on genetic diversity of sMMO due to essential conservation of enzyme function.