CARMIL3 is important for cell migration and morphogenesis during early development in zebrafish.

Cell migration is important during early animal embryogenesis. Cell migration and cell shape are controlled by actin assembly and dynamics, which depend on capping proteins, including the barbed-end heterodimeric actin capping protein (CP). CP activity can be regulated by capping-protein-interacting (CPI) motif proteins, including CARMIL (capping protein Arp2/3 myosin-I linker) family proteins. Previous studies of CARMIL3, one of the three highly conserved CARMIL genes in vertebrates, have largely been limited to cells in culture. Towards understanding CARMIL function during embryogenesis in vivo, we analyzed zebrafish lines carrying mutations of carmil3. Maternal-zygotic mutants showed impaired endodermal migration during gastrulation, along with defects in dorsal forerunner cell (DFC) cluster formation, which affected the morphogenesis of Kupffer's vesicle (KV). Mutant KVs were smaller, contained fewer cells and displayed decreased numbers of cilia, leading to defects in left/right (L/R) patterning with variable penetrance and expressivity. The penetrance and expressivity of the KV phenotype in carmil3 mutants correlated well with the L/R heart positioning defect at the end of embryogenesis. This in vivo animal study of CARMIL3 reveals its new role during morphogenesis of the vertebrate embryo. This role involves migration of endodermal cells and DFCs, along with subsequent morphogenesis of the KV and L/R asymmetry.

Improvement in desulfurization of dibenzothiophene and dibenzothiophene sulfone by Paenibacillus strains using immobilization or nanoparticle coating.

AIMS: Biodesulfurization of fossil fuels is a promising technology for deep desulfurization. Previously, we have shown that Paenibacillus strains 32O-W and 32O-Y can desulfurize dibenzothiophene (DBT) and DBT sulfone (DBTS) effectively. In this work, improvements in DBT and DBTS desulfurization by these strains were investigated through immobilization and nanoparticle coating of cells. METHODS AND RESULTS: Paenibacillus strains 32O-W and 32O-Y immobilized in alginate gel beads or coated with Fe3 O4 magnetite nanoparticles were grown at various concentrations (0.1-2 mmol l-1 ) of DBT or DBTS for 96 h. The production of 2-hydroxybiphenyl (2-HBP) from the 4S pathway biotransformation of DBT or DBTS was measured. The highest amounts of 2-HBP production occurred at concentrations of 0.1 and 0.5 mmol l-1 . Compared to planktonic cultures maximum 2-HBP production increased by 54% for DBT and 90% for DBTS desulfurization with immobilized strains, and 44% for DBT and 66% for DBTS desulfurization by nanoparticle-coated strains. CONCLUSIONS: Nanoparticle-coated and immobilized cells may be of use in efforts to increase the efficiency of biodesulfurization. SIGNIFICANCE AND IMPACT OF THE STUDY: Alginate immobilization or nanoparticle coating of bacterial cells may be useful approaches for the enhancement of biodesulfurization for eventual use on an industrial scale.

Recent trends in bioethanol production from food processing byproducts.

The widespread use of corn starch and sugarcane as sources of sugar for the production of ethanol via fermentation may negatively impact the use of farmland for production of food. Thus, alternative sources of fermentable sugars, particularly from lignocellulosic sources, have been extensively investigated. Another source of fermentable sugars with substantial potential for ethanol production is the waste from the food growing and processing industry. Reviewed here is the use of waste from potato processing, molasses from processing of sugar beets into sugar, whey from cheese production, byproducts of rice and coffee bean processing, and other food processing wastes as sugar sources for fermentation to ethanol. Specific topics discussed include the organisms used for fermentation, strategies, such as co-culturing and cell immobilization, used to improve the fermentation process, and the use of genetic engineering to improve the performance of ethanol producing fermenters.

Biodesulfurization: a model system for microbial physiology research.

Biological desulfurization (biodesulfurization) of dibenzothiophene (DBT) by the 4S pathway is a model system for an enviromentally benign way to lower the sulfur content of petroleum. Despite a large amount of effort the efficiency of the 4S pathway is still too low for a commercial oil biodesulfurization process, but the 4S pathway could potentially be used now for commercial processes to produce surfactants, antibiotics, polythioesters and other chemicals and for the detoxification of some chemical warfare agents. Proteins containing disulfide bonds are resistant to temperature, pH, and solvents, but the production of disulfide-rich proteins in microbial hosts is challenging. The study of the 4S pathway can provide insights as to how to maximize the production of disulfide-rich proteins. Engineering of the operon encoding the 4S pathway to contain a greater content of methionine and cysteine may be able to link use of DBT as a sole sulfur source to increasing 4S pathway activity by increasing the nutritional demand for sulfur. This strategy could result in the development of biocatalysts suitable for use in an oil biodesulfurization process, but the study of the 4S pathway can also lead to a better understanding of microbial physiology to optimize activity of a mult-step co-factor-requiring pathway, as well as the production of highly stable industrially relevant enzymes for numerous applications.

Directed evolution to produce sludge communities with improved oxygen uptake abilities.

Two activated sludge cultures, seeded with activated sludge from the same source, were cultivated for 370 days in synthetic wastewater. Both cultures were transferred weekly to fresh medium; one culture was operated at high dissolved oxygen (DO) (near saturation) and the other at low DO (0.25 mg O2/L). There were significant changes in the abundances of bacterial species and phyla present in each culture throughout the 370-day operational period. In the low DO culture, over time, there was a continuously increasing proportion of cells of species known to encode truncated hemoglobins (Hbs). These are the types of Hbs which may enhance delivery of oxygen to the respiratory chain, to enhance ATP production, especially under low aeration conditions. The levels of heme b, the heme found in Vitreoscilla hemoglobin, increased in parallel to the increase in Hb-encoding species, to much higher levels in the low DO culture than in the high DO culture. Specific oxygen uptake rates increased by 3 % for the high DO culture near the end of the 370-day period, while those for the low DO culture increased steadily to a level 28 % higher than that of the starting culture. Thus, imposition of low DO conditions may, due to selection for Hb-expressing species, be useful in developing bacterial communities with enhanced ability to function efficiently in aerobic wastewater treatment, especially under low aeration conditions.

Recent applications of Vitreoscilla hemoglobin technology in bioproduct synthesis and bioremediation.

Since its first use in 1990 to enhance production of α-amylase in E. coli, engineering of heterologous hosts to express the hemoglobin from the bacterium Vitreoscilla (VHb) has become a widely used strategy to enhance production of a variety of bioproducts, stimulate bioremediation, and increase growth and survival of engineered organisms. The hosts have included a variety of bacteria, yeast, fungi, higher plants, and even animals. The beneficial effects of VHb expression are presumably the result of one or more of its activities. The available evidence indicates that these include oxygen binding and delivery to the respiratory chain and oxygenases, protection against reactive oxygen species, and control of gene expression. In the past 4 to 5 years, the use of this "VHb technology" has continued in a variety of biotechnological applications in a wide range of organisms. These include enhancement of production of an ever wider array of bioproducts, new applications in bioremediation, a possible role in enhancing aerobic waste water treatment, and the potential to enhance growth and survival of both plants and animals of economic importance.

Isolation and characterization of an interactive culture of two Paenibacillus species with moderately thermophilic desulfurization ability.

OBJECTIVE: To isolate and characterize novel thermophilic bacteria capable of biodesulfurization of petroleum. RESULTS: A culture containing two Paenibacillus spp. (denoted "32O-W" and "32O-Y") was isolated by repeated passage of a soil sample at up to 55 °C in medium containing dibenzothiophene (DBT) as sulfur source. Only 32O-Y metabolized DBT, apparently via the 4S pathway; maximum activity occurred from 40 to 45 °C, with some activity up to at least 50 °C. 32O-W enhanced DBT metabolism by 32O-Y (by 22-74 % at 40-50 °C). With sulfate as sulfur source, 32O-Y and 32O-W grew well up to 58 and 63 °C, respectively. Selection of a mixed culture of 32O-Y and 32O-W at 54 °C increased DBT metabolism 36-42 % from 40 to 45 °C. Genome sequencing identified desulfurization gene homologs in the strains consistent with their desulfurization properties. CONCLUSION: The 32O-Y/32O-W culture may be a useful starting point for development of an improved thermophilic petroleum biodesulfurization process.

Characterization of heme protein expressed by ammonia-oxidizing bacteria under low dissolved oxygen conditions.

We have recently reported that expression of an unidentified heme protein is enhanced in a nitrifying activated sludge community under low (0.1 mg O2/L) dissolved oxygen (DO) conditions. A preliminary assessment suggested it may be a type of hemoglobin (Hb) or a lesser-known component of the energy-transducing pathways of ammonia-oxidizing bacteria (AOB) (particularly an oxidase or peroxidase). Here, additional work was done to characterize this protein. Due to the unfeasibility of identifying the protein using gene-based methods, our approach was to carry out assays that target the activity and function of the protein, its location in the cell, and determination of the organisms that express it. Using CO-difference spectra, it was shown that the protein is expressed by AOB preferentially in the cytoplasm, while the pyridine hemochromogen method demonstrated that it has heme c as its prosthetic group. Peroxidase and oxidase assays were carried out on the soluble fraction of the low DO-grown cells; neither the peroxidase nor oxidase activities matched those of the CO-binding heme protein detected. Even though it is not possible to conclusively identify the protein detected as a Hb, all other known possibilities have been ruled out. Further work is needed to verify the identity of the heme protein as a Hb and to determine its type and biochemical role under low oxygen conditions.

Investigation of U.S. landfill GHG reporting program methane emission models.

As part of its commitment to the United Nations Framework Convention on Climate Change, the U.S. annually develops a national estimate of methane emissions from municipal solid waste (MSW) landfills by aggregating activity data from each facility. Since 2010, the U.S. has reported a 20 % decrease in MSW landfill emissions despite a 21 % increase in tons disposed. Operator-submitted data were investigated to understand the causes of this decline. In the U.S., operators of landfills with a gas collection and control system (GCCS) calculate their facility's emissions via two separate approaches - (1) first-order decay (FOD) and (2) collection efficiency assumption (CEA) - and select either result to feed into the annual inventory. The FOD model predicts methane generation proportional to waste disposal and that approach calculated a 19 % increase in total methane generated from 2010 to 2022, whereas generation via the CEA approach decreased by 8.9 %. The amount of measured methane collected has increased 7.5 % for the same years. Discrepancies between the two models' generated methane, assumed gas collection efficiencies, and oxidized methane compound into substantive differences in national estimates. Operators more frequently select the CEA method, which results in decreased national estimates. If only the FOD method was used, U.S. MSW landfill emissions would be 1.3-1.7 times greater than current estimates which is similar to recent extrapolations from remote sensing campaigns in the U.S. Both models contain parameters with large inherent uncertainty. Without measurement methods that continuously quantify both point-source and diffuse emissions, an assessment of either equation's accuracy cannot be made.

Crystallographic structure determination of B10 mutants of Vitreoscilla hemoglobin: role of Tyr29 (B10) in the structure of the ligand-binding site.

Site-directed mutants of the gene encoding wild-type Vitreoscilla hemoglobin were made that changed Tyr29 (B10) of the wild-type Vitreoscilla hemoglobin (VHb) to either Phe or Ala. The wild-type and the two mutant hemoglobins were expressed in Escherichia coli and purified to homogeneity. The binding of the two mutants to CO was essentially identical to that of wild-type VHb as determined by CO-difference spectra. Circular-dichroism spectra also showed the two mutants to be essentially the same as wild-type VHb regarding overall helicity. All three VHbs were crystallized and their structures were determined at resolutions of 1.7-1.9 Å, which are similar to that of the original wild-type structure determination. The Tyr29Phe mutant has a structure that is essentially indistinguishable from that of the wild type. However, the structure of the Tyr29Ala mutant has significant differences from that of the wild type. In addition, for the Tyr29Ala mutant it was possible to determine the positions of most of the residues in the D region, which was disordered in the originally reported structure of wild-type VHb as well as in the wild-type VHb structure reported here. In the Tyr29Ala mutant, the five-membered ring of proline E8 (Pro54) occupies the space occupied by the aromatic ring of Tyr29 in the wild-type structure. These results are discussed in the context of the proposed role of Tyr29 in the structure of the oxygen-binding pocket.

Enhanced heme protein expression by ammonia-oxidizing communities acclimated to low dissolved oxygen conditions.

This study has investigated the acclimation of ammonia-oxidizing communities (AOC) to low dissolved oxygen (DO) concentrations. Under controlled laboratory conditions, two sequencing batch reactors seeded with activated sludge from the same source were operated at high DO (near saturation) and low DO (0.1 mg O2/L) concentrations for a period of 220 days. The results demonstrated stable and complete nitrification at low DO conditions after an acclimation period of approximately 140 days. Acclimation brought about increased specific oxygen uptake rates and enhanced expression of a particular heme protein in the soluble fraction of the cells in the low DO reactor as compared to the high DO reactor. The induced protein was determined not to be any of the enzymes or electron carriers present in the conventional account of ammonia oxidation in ammonia-oxidizing bacteria (AOB). Further research is required to determine the specific nature of the heme protein detected; a preliminary assessment suggests either a type of hemoglobin protein or a lesser-known component of the energy-transducing pathways of AOB. The effect of DO on AOC dynamics was evaluated using the 16S rRNA gene as the basis for phylogenetic comparisons and organism quantification. Ammonium consumption by ammonia-oxidizing archaea and anaerobic ammonia-oxidizing bacteria was ruled out by fluorescent in situ hybridization in both reactors. Even though Nitrosomonas europaea was the dominant AOB lineage in both high and low DO sequencing batch reactors at the end of operation, this enrichment could not be linked in the low DO reactor to acclimation to oxygen-limited conditions.

Trends in the burden and determinants of hypertensive heart disease in the Eastern Mediterranean region, 1990-2019: an analysis of the Global Burden of Disease Study 2019.

Background: Hypertensive heart disease (HHD), one of the end-organ damage consequences of hypertension, is an important public health issue worldwide. Data on the HHD burden in the Eastern Mediterranean region (EMR) are scarce. We aimed to investigate the burden of HHD in the EMR, its member countries, and globally from 1990 to 2019. Methods: We used 2019 Global Burden of Disease (GBD) data to report the HHD age-standardised prevalence, disability adjusted life years (DALYs), years of life lost (YLLs), and mortality, as well as HHD risk factors attribution percent with their 95% uncertainty interval (UI). Global data are reported alongside EMR data, and its 22 respective countries. We compared the burden of HHD by socio-demographic index (SDI), sex, age groups, and countries. Findings: The age-standardised prevalence rate (per 100,000 population) of HHD was higher in the EMR (281.7; 95% UI: 204.5-383.4) in 2019, compared with the global prevalence (233.8; 95% UI: 170.5-312.9). The EMR age-standardised DALYs (per 100,000 population) for HHD in 2019 was 561.9 (361.0-704.1), compared with 268.2 (204.6-298.1) at the global level. There was an increase in HHD prevalence, reduction in mortality, and DALYs between 1990 and 2019 (4.01%, -7.6%, and -6.5%, respectively) in EMR. Among EMR countries, the highest versus lowest rates of age-standardised prevalence, mortality, and DALYs in 2019 [estimate (95% UI)] were in Jordan [561.62 (417.9-747.6)] versus Saudi Arabia [94.9 (69.5-129.0)]; Afghanistan [74.5 (23.7-112.3)] versus Saudi Arabia [4.3 (3.3-5.9)]; and Afghanistan [1374.1 (467.2-2020.7)] versus Qatar [87.11 (64.40-114.29)], respectively. Interpretation: HHD remains a significant problem in the EMR, with a higher burden than global levels. Serious efforts toward high-quality management and prevention are strongly recommended. Based on this study, our recommendation for the EMR is to adopt effective preventive strategies. For example, promoting healthy dietary patterns and prompt screening for undiagnosed HTN in public places, promoting regular blood pressure measurements at home, and creating community awareness about early detection of HTN. Funding: None.

The burden of ischemic heart disease and the epidemiologic transition in the Eastern Mediterranean Region: 1990-2019.

It has been estimated that in the next decade, IHD prevalence, DALYs and deaths will increase more significantly in EMR than in any other region of the world. This study aims to provide a comprehensive description of the trends in the burden of ischemic heart disease (IHD) across the countries of the Eastern Mediterranean Region (EMR) from 1990 to 2019. Data on IHD prevalence, disability-adjusted life years (DALYs), mortality, DALYs attributable to risk factors, healthcare access and quality index (HAQ), and universal health coverage (UHC) were extracted from the Global Burden of Disease (GBD) database for EMR countries. The data were stratified based on the social demographic index (SDI). Information on cardiac rehabilitation was obtained from publications by the International Council of Cardiovascular Prevention and Rehabilitation (ICCPR), and additional country-specific data were obtained through advanced search methods. Age standardization was performed using the direct method, applying the estimated age structure of the global population from 2019. Uncertainty intervals were calculated through 1000 iterations, and the 2.5th and 97.5th percentiles were derived from these calculations. The age-standardized prevalence of IHD in the EMR increased from 5.0% to 5.5% between 1990 and 2019, while it decreased at the global level. In the EMR, the age-standardized rates of IHD mortality and DALYs decreased by 11.4% and 15.4%, respectively, during the study period, although both rates remained higher than the global rates. The burden of IHD was found to be higher in males compared to females. Bahrain exhibited the highest decrease in age-standardized prevalence (-3.7%), mortality (-65.0%), and DALYs (-69.1%) rates among the EMR countries. Conversely, Oman experienced the highest increase in prevalence (14.5%), while Pakistan had the greatest increase in mortality (30.0%) and DALYs (32.0%) rates. The top three risk factors contributing to IHD DALYs in the EMR in 2019 were high systolic blood pressure, high low-density lipoprotein cholesterol, and particulate matter pollution. The trend analysis over the 29-year period (1990-2019) revealed that high fasting plasma glucose (64.0%) and high body mass index (23.4%) exhibited increasing trends as attributed risk factors for IHD DALYs in the EMR. Our findings indicate an increasing trend in the prevalence of IHD and a decrease in mortality and DALYs in the EMR. These results emphasize the need for well-planned prevention and treatment strategies to address the risk factors associated with IHD. It is crucial for the countries in this region to prioritize the development and implementation of programs focused on health promotion, education, prevention, and medical care.

Functional adaptation between yeast actin and its cognate myosin motors.

We employed budding yeast and skeletal muscle actin to examine the contribution of the actin isoform to myosin motor function. While yeast and muscle actin are highly homologous, they exhibit different charge density at their N termini (a proposed myosin-binding interface). Muscle myosin-II actin-activated ATPase activity is significantly higher with muscle versus yeast actin. Whether this reflects inefficiency in the ability of yeast actin to activate myosin is not known. Here we optimized the isolation of two yeast myosins to assess actin function in a homogenous system. Yeast myosin-II (Myo1p) and myosin-V (Myo2p) accommodate the reduced N-terminal charge density of yeast actin, showing greater activity with yeast over muscle actin. Increasing the number of negative charges at the N terminus of yeast actin from two to four (as in muscle) had little effect on yeast myosin activity, while other substitutions of charged residues at the myosin interface of yeast actin reduced activity. Thus, yeast actin functions most effectively with its native myosins, which in part relies on associations mediated by its outer domain. Compared with yeast myosin-II and myosin-V, muscle myosin-II activity was very sensitive to salt. Collectively, our findings suggest differing degrees of reliance on electrostatic interactions during weak actomyosin binding in yeast versus muscle. Our study also highlights the importance of native actin isoforms when considering the function of myosins.

Final Thoughts, Microorganisms Special Issue on Microbial Hemoglobins.

The year 2021 marked the thirty-fifth anniversary of the discovery of microbial hemoglobins by Dale Webster and his colleagues [...].

Redox-mediated interactions of VHb (Vitreoscilla haemoglobin) with OxyR: novel regulation of VHb biosynthesis under oxidative stress.

The bacterial haemoglobin from Vitreoscilla, VHb, displays several unusual properties that are unique among the globin family. When the gene encoding VHb, vgb, is expressed from its natural promoter in either Vitreoscilla or Escherichia coli, the level of VHb increases more than 50-fold under hypoxic conditions and decreases significantly during oxidative stress, suggesting similar functioning of the vgb promoter in both organisms. In the present study we show that expression of VHb in E. coli induced the antioxidant genes katG (catalase-peroxidase G) and sodA (superoxide dismutase A) and conferred significant protection from oxidative stress. In contrast, when vgb was expressed in an oxyR mutant of E. coli, VHb levels increased and the strain showed high sensitivity to oxidative stress without induction of antioxidant genes; this indicates the involvement of the oxidative stress regulator OxyR in mediating the protective effect of VHb under oxidative stress. A putative OxyR-binding site was identified within the vgb promoter and a gel-shift assay confirmed its interaction with oxidized OxyR, an interaction which was disrupted by the reduced form of the transcriptional activator Fnr (fumurate and nitrate reductase). This suggested that the redox state of OxyR and Fnr modulates their interaction with the vgb promoter. VHb associated with reduced OxyR in two-hybrid screen experiments and in vitro, converting it into an oxidized state in the presence of NADH, a condition where VHb is known to generate H2O2. These observations unveil a novel mechanism by which VHb may transmit signals to OxyR to autoregulate its own biosynthesis, simultaneously activating oxidative stress functions. The activation of OxyR via VHb, reported in the present paper for the first time, suggests the involvement of VHb in transcriptional control of many other genes as well.

Recent advances in understanding the structure, function, and biotechnological usefulness of the hemoglobin from the bacterium Vitreoscilla.

The hemoglobin from the bacterium Vitreoscilla (VHb) is the first microbial hemoglobin that was conclusively identified as such (in 1986). It has been extensively studied with respect to its ligand binding properties and mechanisms, structure, biochemical functions, and the mechanisms by which its expression is controlled. In addition, cloning of its gene (vgb) into a variety of heterologous hosts has proved that its expression results substantial increases in production of a variety of useful products and ability to degrade potentially harmful compounds. Recent studies (since 2005) have added significant knowledge to all of these areas and shown the broad range of biotechnological applications in which VHb can have a positive effect.

The Discovery of Vitreoscilla Hemoglobin and Early Studies on Its Biochemical Functions, the Control of Its Expression, and Its Use in Practical Applications.

In 1986, the surprising identification of a hemoglobin (VHb) in the bacterium Vitreoscilla greatly extended the range of taxa in which this oxygen binding protein functions. Elucidation of many of its biochemical properties and relation to overall cell physiology, as well as the sequence of the gene encoding it and aspects of control of its expression were determined in the following years. In addition, during the early years following its discovery, strategies were developed to use its expression in heterologous microbial hosts to enhance processes of practical usefulness. The VHb discovery also served as the foundation for what has become the fascinatingly rich field of bacterial hemoglobins. VHb's position as the first known bacterial hemoglobin and its extensive use in biotechnological applications, which continue today, make a review of the early studies of its properties and uses an appropriate and interesting topic thirty-five years after its discovery.

Engineering of ethanolic E. coli with the Vitreoscilla hemoglobin gene enhances ethanol production from both glucose and xylose.

Escherichia coli strain FBR5, which has been engineered to direct fermentation of sugars to ethanol, was further engineered, using three different constructs, to contain and express the Vitreoscilla hemoglobin gene (vgb). The three resulting strains expressed Vitreoscilla hemoglobin (VHb) at various levels, and the production of ethanol was inversely proportional to the VHb level. High levels of VHb were correlated with an inhibition of ethanol production; however, the strain (TS3) with the lowest VHb expression (approximately the normal induced level in Vitreoscilla) produced, under microaerobic conditions in shake flasks, more ethanol than the parental strain (FBR5) with glucose, xylose, or corn stover hydrolysate as the predominant carbon source. Ethanol production was dependent on growth conditions, but increases were as high as 30%, 119%, and 59% for glucose, xylose, and corn stover hydrolysate, respectively. Only in the case of glucose, however, was the theoretical yield of ethanol by TS3 greater than that achieved by others with FBR5 grown under more closely controlled conditions. TS3 had no advantage over FBR5 regarding ethanol production from arabinose. In 2 L fermentors, TS3 produced about 10% and 15% more ethanol than FBR5 for growth on glucose and xylose, respectively. The results suggest that engineering of microorganisms with vgb/VHb could be of significant use in enhancing biological production of ethanol.