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PRELIMINARY MECHANISMS FOR THE FORMATION OF MULTISPECIES BIOFILMS
MICROBIAL INTERACTIONS AMONG MULTISPECIES BIOFILMS
PRELIMINARY MECHANISMS FOR THE RESISTANCE OF MULTISPECIES BIOFILMS TO DISINFECTANTS
CONCLUSIONS AND FUTURE PERSPECTIVES
review article| July 29 2021
Formation of Multispecies Biofilms and Their Resistance to Disinfectants in Food Processing Environments: A Review
QUN LI;
QUN LI
1College of Food Science and Technology, Huazhong Agriculture University, Wuhan, Hubei 430070, People's Republic of China
LING LIU;
LING LIU
AILING GUO;
AILING GUO *
2National Research and Development Center for Egg Processing, Wuhan, Hubei 430070, People's Republic of China
*Author for correspondence. Tel: +86 15342241896; Fax: 027-87384670; E-mail: ailingguo234@163.com.
XINSHUAI ZHANG;
XINSHUAI ZHANG
WUKANG LIU;
WUKANG LIU
YAO RUAN
J Food Prot (2021) 84 (12): 2071–2083.
https://doi.org/10.4315/JFP-21-071
QUN LI, LING LIU, AILING GUO, XINSHUAI ZHANG, WUKANG LIU, YAO RUAN; Formation of Multispecies Biofilms and Their Resistance to Disinfectants in Food Processing Environments: A Review. J Food Prot 1 December 2021; 84 (12): 2071–2083. doi: https://doi.org/10.4315/JFP-21-071
In food processing environments, various microorganisms can adhere and aggregate on the surface of equipment, resulting in the formation of multispecies biofilms. Complex interactions among microorganisms may affect the formation of multispecies biofilms and resistance to disinfectants, which are food safety and quality concerns. This article reviews the various interactions among microorganisms in multispecies biofilms, including competitive, cooperative, and neutral interactions. Then, the preliminary mechanisms underlying the formation of multispecies biofilms are discussed in relation to factors, such as quorum-sensing signal molecules, extracellular polymeric substances, and biofilm-regulated genes. Finally, the resistance mechanisms of common contaminating microorganisms to disinfectants in food processing environments are also summarized. This review is expected to facilitate a better understanding of interspecies interactions and provide some implications for the control of multispecies biofilms in food processing.
Multispecies interaction of biofilms is often cooperative, competitive, or neutral.
QS molecules, EPS, and biofilm-regulated genes affect multispecies biofilm formation.
Disinfectant resistance of multispecies biofilms depends on EPS and biofilm age.
Studies of multispecies biofilms are less focused on high-diversity biofilms.
Disinfectants, Food processing environments, Interactions, Mechanisms, Multispecies biofilms
To adapt to various stress conditions imposed by the environment, microorganisms have developed different survival strategies, including biofilm formation. Biofilms are a community of microorganisms encapsulated in extracellular polymeric substances (EPS), mainly composed of polysaccharides, proteins, lipids, and nucleic acids (15). About 40 to 80% of bacteria on the Earth can form biofilms, just as some fungi (22, 27, 37). However, most biofilms found in natural, clinical, or industrial environments are formed by two or more species of microorganisms instead of a single species and are, thus, called multispecies biofilms (25).
Due to the species diversity present in the raw materials, multispecies biofilms are often formed on food contact surfaces (e.g., cutters, spiral conveyors, and related equipment) and nonfood contact surfaces (e.g., drains and pipes) in the meat and dairy industry (75, 103). Even after normal hygiene and disinfection procedures, several microorganisms can still remain in food production environments, such as Staphylococcus, Stenotrophomonas, Streptococcus, Pseudomonas, Acinetobacter, and Listeria (24, 34, 53, 65). These microorganisms may affect food quality and safety, because they not only coexist in biofilms but also persist in production environments, posing continual and potential contamination risks during food manufacture.
Microscopic techniques have helped reveal three types of bacterial organization in multispecies biofilms: microcolonies, which form separate small niches in parallel; layered structures, in which each species is located in a different layer; and multispecies mixing patterns (35, 61, 74). The distribution of these structures largely depends on the type of microorganisms and leads to competitive and cooperative or neutral interactions (3). These interactions bring about some ecological advantages for microorganisms, such as the availability of nutrients and resistance to harsh environments and disinfectants. Biofilm formation enhances the persistence of internal microorganisms inside the biofilm matrix. This persistence leads to an increase of microbial load in the food processing environment and in the subsequent food products, causing food spoilage, shortened shelf life, and increased risk of infectious diseases originating from food sources (16). For example, in the food industry, pathogenic bacteria can form biofilms in processing facilities, causing corrosion of facility surfaces and blocking of pipes (28). Aeromonas and Pseudomonas are important bacteria associated with the spoilage of refrigerated freshwater fish. The coculture of these two bacteria could significantly increase the maximum growth rate, promote biofilm formation, and improve the spoilage capacity of bacterial strains, resulting in spoilage of grass carp (113).
Competition, cooperation, and communication of microorganisms in food processing environments lead to the complex behavior of multispecies biofilms, enhancing the infectivity and resistance of microorganisms in the biofilms (93). An increase in disinfectant resistance will significantly reduce the efficiency of cleaning and disinfection processes, allowing microorganisms to remain in food and food processing equipment (97). Microorganisms that form multispecies biofilms may be affected by other microorganisms, thus changing the survival status as a potential source of contamination. The multispecies biofilms formed by Lactobacillus plantarum and Listeria innocua can change from an almost completely unculturable state to a culturable state, representing a potential source of contamination (76).
Therefore, the formation of multispecies biofilms can give rise to the spread and persistence of harmful microorganisms in food processing environments, shorten the shelf life of food, and increase the probability of human illness after ingestion of contaminated food. Therefore, special research attention should be paid to the various potential negative effects of multispecies biofilms. This article will discuss these aspects from three perspectives: the preliminary mechanisms for the formation of multispecies biofilms, the interactions among the microorganisms in multispecies biofilms, and the preliminary mechanisms for the resistance of microorganisms in multispecies biofilm to disinfectants. From the perspective of food safety, this review will contribute to more accurate risk analysis and the development of effective strategies for the prevention and control of harmful multispecies biofilms.
Research efforts have revealed that the formation process of multispecies biofilms consists of five successive steps: (i) reversible attachment; (ii) irreversible attachment; (iii) microcolony formation; (iv) biofilm maturation; and (v) dispersion or separation (Fig. 1). The formation of bacterial biofilms starts with the formation of an appropriate surface layer after the absorption of organic or inorganic molecules (such as polysaccharides, proteins, lipids, and fatty acids) and then single or mixed communities are embedded into heterostructured EPS (16). Once bacteria attach to biological or abiotic surfaces, they communicate with each other by using an extracellular signaling system on the basis of quorum sensing (QS) (71). In the whole formation process of multispecies biofilms, biofilm-regulated genes will be activated and play corresponding roles. In addition, biofilm formation is a process in which microorganisms change phenotypes for adaptation to environmental stress or immune response. Multispecies interactions increase the probability of changes in biofilm-regulated genes. Therefore, the formation mechanisms of multispecies biofilms are closely related to QS, EPS, biofilm-regulated genes, and other factors (Fig. 2).
Hypothetical processes of multispecies biofilm formation: (1) reversible attachment, planktonic cells attach nonspecifically to the surface and aggregate loosely, which can dissociate and revert to the form of planktonic cells; (2) irreversible attachment, microbial cells irreversibly attach to the surface, the cells begin to secrete EPS and QS factors, and the aggregated population continues to grow; (3) microcolony formation, microbial cells interact to form microcolonies, and both interspecies and intraspecies cells communicate through QS factors and EPS; (4) biofilm maturation, microcolonies continue to grow and form mature biofilm structures; (5) dispersion or separation: (a) the microbial cells diffuse actively and return to the planktonic states; (b) changes in environmental conditions, such as cleaning and disinfection measures, passively detach biofilm aggregates from mature biofilms (50, 70). The spatial arrangement of microorganisms in the multispecies biofilms in this figure is based on the mixed model (61).
Preliminary mechanisms for the formation of multispecies biofilms: (A) QS signal molecules; (B) EPS; (C) biofilm-regulated genes; and (D) others.
QS signal molecules
QS is a way in which species sense cell density and, accordingly, regulate gene expression (7). There has been increasing evidence showing that multiple microorganisms can sense specific QS signal molecules, enabling them to perceive and react with other microorganisms nearby (20). QS plays a key role in the collective behavior of bacteria, including the initial stage of biofilm attachment and the maturation or diffusion of biofilms at the intraspecies and interspecies levels (87). QS molecules produced by bacteria are called autoinducers (AI), used for one-way, two-way, or multiway communication (85). When the concentration of AI reaches the threshold value, the expression of certain genes will be changed (108). In general, AI are involved in biofilm formation and microorganism motility and also coordinate intra- and interspecies interactions (32, 48). Although fungi have not been shown to produce the analogs of bacterial AI, QS signal molecules, such as farnesol, tyrosol, phenylethanol, and tryptophol, have been identified in fungi (Fig. 2A) (110). Currently, the importance of some QS signal molecules in multispecies biofilms has been gradually revealed, such as acyl-homoserine lactones (AHL) and autoinducer-2 (AI-2; Fig. 2A).
AHL-mediated QS is a vital regulatory mechanism for the formation and structure of gram-negative bacterial biofilms, regulating the maturation of biofilms (52). Chandler et al. (12) reported that AHL is indispensable for interspecies competition. Zhu et al. (114) found that there are competitive interactions between Pseudomonas fluorescens and Shewanella baltica, and S. baltica may inhibit the production of AHL in dual-species biofilms or consume the AHL secreted by P. fluorescens. de Almeida et al. (17) revealed that Salmonella Enteritidis can form stronger biofilms in the presence of AHL from other species.
AI-2, a class of cyclic oligopeptides, is a nonspecific QS signal molecules produced by both gram-negative and gram-positive bacteria (98). It has been proved that S-ribosyl homocysteine is a precursor of AI-2, playing an indispensable role in the biofilm formation of Listeria monocytogenes (8). Similarly, the QS system of Staphylococcus aureus seems to be involved in the regulation of L. monocytogenes attachment and biofilm formation in an early stage (91). Laganenka and Sourjik (51) have established the importance of the AI-2–mediated signal in mixed-species biofilm formation of Enterococcus faecalis and Escherichia coli; lsr encodes the LSR protein. AI-2 accumulates in the extracellular medium with early growth, and once a certain cell density is reached, these levels are depleted by import via the Lsr transport system. In the dual-species biofilms, E. faecalis could secrete extra AI-2 to enhance the expression of E. coli lsr, which promotes the coaggregation of E. coli and E. faecalis, increasing the stress resistance of both E. coli and E. faecalis. Thus, another putative function for the Lsr is interference in AI-2–dependent QS. The interaction between P. fluorescens and S. aureus in dual-species biofilms could promote the production of extracellular polysaccharides regulated by AI-2 and result in the complex biofilm structures (107). Pseudomonas aeruginosa and P. fluorescens were found to significantly inhibit the biofilm formation of Aeromonas hydrophila on crab coupons and significantly reduce the levels of AHL and AI-2, supporting the relationship between QS and biofilm formation (43).
QS is an indispensable mechanism on the basis of the production, secretion, and subsequent concentration-dependent reactions of the signal molecule AI (41). Interference with QS can help inhibit the formation of biofilms by foodborne pathogens and reduce possible disease outbreaks. In the future, it will be necessary to study QS in the coculture of multiple microorganisms.
EPS are composed of a series of polymers, such as carbohydrates, proteins, extracellular DNA, and extracellular RNA, which interact with and transport small molecules such as QS signals and redox shuttles to coordinate the activity of the whole biofilm matrix (90). The existence of EPS not only contributes to the establishment of biofilms but also promotes growth, including mediating cell attachment and expanding the volume of cell clusters (46, 68). The expansion of cell cluster volume allows the entry of EPS-producing strains into the nutrient-rich area in the biofilms, while those strains that do not produce EPS are at a disadvantage. EPS-producing strains and non–EPS-producing strains will compete for nutrients and spaces, with the former generally having a stronger biofilm formation ability, and thus a higher possibility to gain a foothold in multispecies biofilms (106). In the formation of biofilms by two or more microbial strains, the interactions among different EPS components may change the overall composition of EPS.
EPS of microbial communities can be divided into two categories: (i) the components related to the cell surface, such as fimbriae, flagella, and functional amyloids that regulate bacterial attachment, stability, and autoimmune response and (ii) extracellular secretion, such as bacterial extracellular polysaccharides, proteins, extracellular DNA and extracellular RNA (Fig. 2B) (45). Bacterial EPS components, such as curli fimbriae, cellulose, capsular polysaccharides, and lipopolysaccharides, have been reported to not only improve the biofilm formation ability of bacteria but also promote the viability and resistance of biofilms (104). The structure of the multispecies biofilms formed by Salmonella Typhimurium and E. coli O157:H7 strains is highly dependent on the characteristics of the coexisting strains, particularly EPS components, including curli fimbriae and extracellular polysaccharide cellulose (106). The bacteria that have EPS and fimbriae have an advantage over those types of bacteria that do not produce these components. However, those types of bacteria that lack these components can still form mixed biofilms by combining with types of bacteria that do have these features, which is likely to improve the persistence in the biofilm of both types of bacteria (73). Curli fimbriae endow E. coli O157:H7 with competitiveness in multispecies biofilms, possibly due to the interactions between Shiga toxin–producing E. coli and some spinach-associated microorganisms (10). The interaction between EPS protein TasA produced by Bacillus subtilis and extracellular polysaccharides produced by Streptococcus mutans plays a crucial role in the initial attachment of bacteria during the formation of dual-species biofilms (23). During the processing of ultrahigh-temperature-sterilized milk, the poor biofilm-forming bacterium Lactococcus lactis would benefit from the enhanced attachment provided by the matrix of P. fluorescens, and in return, P. fluorescens would use the metabolites produced by L. lactis as a nutrient source (47). L. plantarum and yeast coexist in a wide range of fermented foods, and the ability of L. plantarum to adhere to surfaces with yeast cells enables the formation and maintenance of biofilms, and cell-to-cell attachment may occur through the interactions between a lectinlike protein of L. plantarum and mannan of yeast cells (37). Pseudomonas and Basidiobolus coexist in biofilms formed in the drinking water distribution system, and the extracellular enzymes secreted by the Basidiobolus can degrade high-molecular-weight compounds and release secondary metabolites, used by other microorganisms for growth (22).
The coexistence of different species or even different communities makes the analysis of the composition and biological characteristics of EPS more complex. Increasing details of EPS components in multispecies biofilms are being revealed; however, there is still very limited information about the functions and structures and how different microorganisms regulate the production and interaction of EPS components within biofilms. In addition to providing structural stabilities and functional environments, EPS also enhance the tolerance of biofilms to antimicrobials and disinfectants. Therefore, understanding the changes in the chemical composition of EPS in multispecies biofilms will help to exploit the weaknesses of pathogenic biofilms. Besides, an in-depth understanding of the various properties of biofilm EPS can facilitate the development of effective methods for controlling biofilm-related contamination.
Biofilm-regulated genes
Regulatory genes are expressed at each stage of biofilm formation. The development of biofilms involves the regulation of hundreds of biofilm-specific genes, including those related to QS, EPS, metabolism, stress response, and transport (Fig. 2C) (21). Compared with the growth of individual species, some genetic characteristics affecting biofilm development will change, depending on the interaction among specific species (94). If there are genotypic differences in the expression of adhesin in multispecies biofilms, the strains with low adhesion may become increasingly dominant, because they can compete for more nutrients from highly adherent cells. As flagella genes of L. monocytogenes, degU, flaA, and motB play indispensable roles in controlling bacterial adhesion and regulating biofilm formation. flaA gene codes for flagellin, the principal component of bacterial flagellum, affecting the motility of strains (11). The MotB proteins, which form the stator component of the flagellum itself, serve a function in transmembrane transport of protons in the aforementioned flagellar motor complex (11). Strains with strong motility have weak adhesion, which is not conducive to the biofilm formation. DegU was shown to be essential for flagellar synthesis and bacterial motility in L. monocytogenes, and gradual increases in DegU phosphorylation levels are critical in the transition from motile to sessile biofilm-forming cells (33). Compared with single-species biofilms, these biofilm-related genes in the dual-species biofilms formed by Vibrio parahaemolyticus and L. monocytogenes were down-regulated, which may be a vital reason for the decreases in the biomass, biovolume, and thickness of the dual-species biofilms (13). During the biofilm development of Trichoderma viride and Azotobacter chroococcum, the attachment of T. viride to the mycelium would lead to significant changes in transcriptome, especially the expression of genes related to biofilm formation, including the genes encoding ATP-binding cassette (ABC) transporters, RNA-dependent RNA polymerase, translation elongation factor EF-1, dual homeobox 4, and molecular chaperones (100).
Horizontal gene transfer, the movement of genetic material between microorganisms, frequently occurs in microbial communities (44). Haubert et al. (36) demonstrated a transfer of the resistance gene tetM from L. monocytogenes to E. faecalis in Minas frescal cheese. Horizontal gene transfer can enable microorganisms to share genetic elements with other cells and is, therefore, associated with biofilm formation (63). Diani et al. (19) studied Enterococcus faecium and E. faecalis that are often detected in fermented sausages to understand the relationship between the presence of the esp gene and biofilm formation. As a result, the presence of the esp gene was found to promote the biofilm formation of Enterococci. Horizontal transfer of the esp gene may lead to the spread of biofilms, not only between Enterococcus strains, but also between different species, such as Salmonella. At present, there are still very few reports on the promotion of biofilm formation by horizontal gene transfer; this is worthy of further research.
The formation of multispecies biofilms is related to the expression of regulatory genes of microorganisms in the biofilms, which is of great significance for understanding the adaptation and subsequent evolution of microbial characteristics. At present, there are still a number of gaps in these areas. In the future, more attention should be paid to these issues to improve our understanding of the mechanisms for multispecies biofilm formation.
Environmental adaptability, colonization sequence, presence of antibacterial substances, substrates, or colonization surfaces of microorganisms also have great impact on the formation of multispecies biofilms (Fig. 2D). An increase in biofilm formation may be a common adaptive response for long-term coexistence. Enhanced biofilm formation is common for cocultured bacteria coexisting in the original environment. Reduction in biofilm formation will be even more pronounced when the cocultured bacteria are from environments without long-term coexistence of bacteria, indicating that the induction of interspecies biofilms is, at least partly, due to previous contacts and the formation of interspecies biofilms is an adaptive response (64). It is possible for microorganisms to recontact surfaces to form biofilms in the presence of preexisting biofilms formed by symbiotic or spoilage bacteria or other pathogens. Therefore, colonization sequence may play a key role in determining the composition and dominant species of multispecies biofilms. In the formation process of multispecies biofilms, early precolonization could significantly affect the competition between E. coli O157:H7 and E. coli O111:H8, and the growth of the precolonized colonies could exceed that of the latter colonized colonies (105). For example, after the precolonization of L. monocytogenes, the dual-species biofilms formed with P. fluorescens showed flat and thin-layer structures, while the precolonization of P. fluorescens would result in a multilayer structure with a higher thickness and roughness (83). Pang and Yuk (84) later found that colonization sequence does not affect populations of the dual-species biofilms of L. monocytogenes and P. fluorescens but influence the attachment of the dual-species biofilms. The result indicates that colonization sequence affects the structures and adhesion of the biofilms formed by L. monocytogenes and P. fluorescens but has no significant effect on cell density.
The formation mechanisms of multispecies biofilms vary among different surfaces or substrates. For different dairy substrates (brain heart infusion, cow's milk, and whey protein), Enterococcus faecalis was always dominant in multispecies biofilms, while other pathogens, including L. monocytogenes, S. aureus, and Bacillus cereus, could not form biofilms in a multispecies system with whey protein as the substrate, and only adhesion was observed (4). Iniguez-Moreno et al. (38) studied the formation of dual-species biofilms of S. aureus 4E and Salmonella and found that biofilms have better adhesion on stainless steel surfaces than on polypropylene, which may be related to the improvement of iron use. Some microorganisms may release substances that inhibit biofilm formation. Some species of Bacillus are producers of antibacterial compounds (peptides, lipopeptides, bacteriocins, and inhibitory substances similar to bacteriocins) against bacteria and fungi (6). For example, B. cereus RC6 secretes two enzymes that degrade casein and show antibacterial activity against L. monocytogenes (77). Staphylococcus xylosus released a molecule with a weight greater than 30 kDa in its growth supernatant, which can resist proteolytic enzymes and inhibit the biofilm formation of S. aureus MW2 (56). P. aeruginosa inhibits the growth of S. aureus or can even inactivate S. aureus cells, due to the production of several toxic compounds, including hydrogen cyanide, pyocyanin, and alkyl-hydroxyquinoline N-oxide, which can block the electron transport chain of S. aureus (66).
Due to the complexity of microbial interactions, it is unreasonable to predict the formation of multispecies biofilms just on the basis of one single mechanism. The combination of QS, EPS, biofilm-regulated genes, environment, and other factors ultimately affects the behavior of microorganisms. For example, QS is highly correlated with EPS, which affects the function of biofilms. In the biofilms of P. aeruginosa, extracellular polysaccharides promote the absorption of QS signal molecules in the biofilms, and the biofilm matrix activates or inactivates the QS process (5). Compared with single-species biofilms, the dual-species biofilms formed by V. parahaemolyticus and L. monocytogenes exhibited significantly lower biomass and biological volume and thickness. These differences were ascribed to the number of bacteria in dual-species biofilms, expression of biofilm-regulated genes, metabolic activity, extracellular polysaccharides, and proteins (13).
Interspecies interactions affect the biofilm phenotypes of bacteria. In recent years, there have been increasing studies of the interactions between major foodborne pathogens and other food-related microorganisms or microbial community members residing in food processing environments in multispecies biofilms. The interactions among multiple species are generally cooperative, competitive, or neutral and thus determine the dominant species in biofilms (Table 1).
Examples of microbial interactions among multispecies biofilms
Competitive interactions
In food processing environments, competitive interactions occur with one or more species at a disadvantage in coculturing. For instance, P. aeruginosa and S. aureus coexist in many biofilm-related infections and have a relationship that is competitive in nature (72). When cocultured with bacteria (Citrobacter, Hafnia, Aeromonas, and Carnobacterium) on meat processing surfaces, Salmonella Typhimurium and E. coli O157:H7 can be harmoniously integrated into competitive biofilms, while facultative anaerobes may have a competitive advantage over strict anaerobes in establishing multispecies biofilms (101).
The phenotypes involved in microbial competition in a multispecies biofilm may have developed to aid in competition for resources, such as space, nutrients, and energy through means of rapid and efficient growth, production of nutrient scavenging molecules, superior position in the niches, direct competition with other species by producing bacteriostatic substances, or through contact-dependent inhibition (46, 62, 88, 92, 96). Vibrio cholerae and E. coli have an antagonistic effect on the dual-species biofilms at the air-liquid interfaces, in which V. cholerae plays a dominant role in the viscous elasticity of the biofilms, and forms mature biofilms 18 h earlier than E. coli (1). In the multispecies biofilms containing L. monocytogenes or S. aureus, the number of sessile cells in each member of relevant microbial communities, including pathogens, is smaller than that in single- or dual-species biofilms, which may be ascribed to competition for nutrients (78). The competitive interaction between Salmonella and P. aeruginosa would result in a decrease in the density of dual-species biofilms on stainless steel coupons, which may be caused by AHL, a class of signal molecules produced by P. aeruginosa and reported to inhibit the Salmonella biofilm (81). The indole produced by E. coli can help resist the AHL-producing gram-negative bacteria and enhance its competitiveness in biofilms (89). The dual-species biofilms formed by L. monocytogenes and B. cereus isolated from dairy products exhibited competitive interactions as well, which is dependent on the ability of B. cereus to produce antibacterial metabolites against L. monocytogenes (3). However, competitive interactions are not always deleterious to microorganisms. For example, competition is beneficial when the coexisting species have overlapping metabolic niches (29). Moreover, competitive interactions enhance the resistance of dominant bacteria to disinfectants in multispecies biofilms, conductive to the survival of biofilms (49).
Cooperative interactions
Madsen et al. (64) proposed the notion of cooperative interacting microorganisms, considering that the total amount of multispecies biofilms was higher than the total amount of all single-species biofilms, and found that cooperative interactions account for 13% of the interactions among microorganisms in fresh water, ocean, and soil communities. Cooperative interactions may benefit single or multiple sides of microorganisms at the same time, which is conducive to the formation of biofilms. Ralstonia insidiosa and Burkholderia caryophylli isolated from fresh-cut processing facilities exhibited a synergy relationship with E. coli, and E. coli benefited from the cooperative interactions (60). A typical feature of cooperative interactions in microbial communities is that the partner usually excretes metabolites with a positive impact on the growth of the other microorganism (99, 112). Microorganisms may excrete metabolites through cell leakage or as by-products or end products of their own metabolism, which are absorbed by other microorganisms (57, 69). It has been reported that there were cooperative interactions in the growth of multispecies biofilms of P. fluorescens, L. plantarum, and Leuconostoc pseudomesenteroides, possibly because P. fluorescens produced extra EPS, which was absorbed by other microorganisms (54). The mixed culture of L. monocytogenes and R. insidiosa also displayed synergistic interactions, with the generation of a large amount of extra EPS in the dual-species biofilms, making the biofilms more robust than any single-species biofilms (58). Overall, cooperative interactions among microorganism in biofilms tend to promote adhesion, growth, and disinfectant resistance (3).
Other interactions
When multiple species coexist in the same biofilm environments, there may be a neutral state of noninterference with each other, which only happens in rare cases (109). For example, the presence of E. coli or its metabolites in the pretreatment medium did not affect the biofilm formation of L. monocytogenes (18). Salmonella Typhimurium, L. monocytogenes, and Pectobacterium carotovorum did not compete with A. hydrophila; instead, they showed neutral interactions with A. hydrophila in multispecies biofilms (43). Agustín and Brugnoni (2) simulated the interactions between Listeria and resident yeasts in the juice processing environment. As a result, the number of L. innocua cells increased significantly in the presence of Candida tropicalis and Candida krusei and decreased significantly in the presence of Rhodotorula mucilaginosa and Candida kefyr. When cocultured with C. tropicalis, L. monocytogenes also decreased significantly in the number of cells. These results reveal the synergistic or antagonistic interactions between Listeria and resident yeasts; therefore, cooperative and competitive interactions may exist simultaneously among multiple species.
The interactions among microorganisms in multispecies biofilms are affected by the environment and their own properties. Competitive interactions are more common than cooperative and neutral interactions under conditions of limited nutrition and space (41). The cooperative and competitive interactions will largely determine the dominant species in the mixed biofilms, thus determining the structure and activity of the multispecies community. Therefore, in-depth exploration of microbial interactions in multispecies biofilms is highly necessary for understanding the interactions in multispecies biofilms and how these interactions affect biofilm resistance to disinfectants, which may help avoid the formation of harmful microbial biofilms and promote that of desired microbial biofilms.
Increasing evidence has indicated that the interactions among different species can greatly increase the resistance of multispecies biofilms to biocides (95). Compared with single-species biofilms, multispecies biofilms are characterized by greater resistance to disinfectants, such as chlorine, benzalkonium chloride, sodium hypochlorite, peracetic acid, quaternary ammonium compounds (QAC), and hydrogen peroxide (Table 2). The studies concerning the resistance of major foodborne pathogens to some industrial disinfectants have also revealed that disinfectants cannot completely inactivate target microorganisms in multispecies biofilms (14, 26). However, the mechanisms underlying the resistance of multispecies biofilms are not fully understood yet. However, some factors potentially associated with the increase in resistance of microorganisms to disinfectants in multispecies biofilms have been proposed, such as the type of strains, EPS formation, biofilm age, environment, and spatial distribution of strains.
Examples of multispecies biofilms with enhanced disinfectant resistance
Type of strains
In most cases, multispecies biofilms are more resistant to disinfection than single-species biofilms, and dual-species biofilms with foodborne pathogens are more resistant to disinfection treatment than single-species biofilms, as well (49). For example, the presence of P. fluorescens in the dual-species biofilms could protect L. monocytogenes cells against disinfectant treatments (83). When E. coli and E. faecalis were cocultured to form biofilms, the disinfectant resistance of both species was enhanced, and the coaggregation in the biofilms also greatly improved the survival rate of E. faecalis, because the E. faecalis cells in the biofilms were covered by small colonies of E. coli (51). Oxaran et al. (78) established mature multispecies biofilms containing L. monocytogenes and Staphylococcus aureus on stainless steel coupons and treated them with peracetic acid. As a result, S. aureus in multispecies biofilms has a bigger number of sessile cells than that in single-species biofilms, indicating that some specific microorganisms in multispecies biofilms can provide certain protection on other microorganisms. This may be due to the presence of specific microorganisms that affect the composition of the biofilm matrix and are more resistant to disinfectants.
However, the interactions among different strains in multispecies biofilms may also have no positive impact on the disinfection resistance of microorganisms. Gomes et al. (31) isolated two pathogens from drinking water, namely, Acinetobacter calcoaceticus and Stenotrophomonas maltophilia, and found that the dual-species biofilms formed by the two microorganisms on polyvinyl chloride surfaces were more sensitive to sodium hypochlorite than single-species biofilms. In the dual-species biofilms formed by Salmonella Typhimurium and S. aureus, Gkana et al. (30) found that the interspecies interactions had no significant effect on the overall disinfectant tolerance (benzalkonium chloride, sodium hypochlorite, and peracetic acid) of the whole species. In addition, Iniguez-Moreno et al. (38) found that the interspecies interactions between S. aureus 4E and Salmonella had a negative effect on the antimicrobial resistance of each microorganism, compared with the single-species biofilms. These findings suggest that the increase in the resistance of multispecies biofilms to disinfectants is not a universal phenomenon.
As a physical barrier, EPS limit the penetration of disinfectants or the interactions of microorganism with disinfectants, thus hindering the function of disinfectants (9). In multispecies biofilms, EPS produced by one species may protect another species from biocides by limiting the diffusion process (95). For example, previous studies have demonstrated the critical role of EPS in protecting the multispecies biofilms of Salmonella Typhimurium and culturable native microorganisms in lettuce from UV-C radiation (42). P. aeruginosa was found to inhibit the growth of Salmonella cells in biofilms but protect Salmonella cells in biofilms from chlorine or Ecolab Whisper V by providing more EPS in dual-species biofilms than in single-species biofilms (81). P. fluorescens could produce more polysaccharides in dual-species biofilms, which have a protective effect on Salmonella Enteritidis, thereby enhancing the resistance of Salmonella Enteritidis to QAC (79). The interaction between P. fluorescens and S. aureus in the dual-species biofilms could promote the production of extracellular polysaccharides, which may contribute to an increase in the resistance of the communities to carvacrol (107). In the dual-species biofilms formed on stainless steel coupons in chicken juice, Salmonella Enteritidis showed higher chlorine resistance, which may be due to the presence of more glycoconjugates in the dual-species biofilms to hinder the effect of chlorine (82).
Biofilm age
The signals and metabolites may be different between the microorganisms in biofilms and the planktonic state, which may cause different physiological changes in neighboring cells. Therefore, the resistance of microorganisms to disinfectants in multispecies biofilms is related to the biofilm age as well. Mature biofilms are generally more tolerant to stress conditions than freshly formed biofilms due to the strong three-dimensional structure of adherent layers of bacterial cells, which constitute a physical barrier that restricts and blocks the penetration of disinfectants or other chemicals (104). For example, the formation of biofilms with preexisting P. fluorescens on stainless steel enhanced the resistance of L. monocytogenes to disinfectants, though the setting reduced the transfer rate of L. monocytogenes to salmon slices (84). Most of the spoilage bacteria in the microbrewery environment showed strong biofilm-forming ability on polystyrene and stainless steel, while the mature biofilm on stainless steel was not sufficiently inactivated by peracetic acid (67). Therefore, in food processing environments, cleaning and sanitation of equipment and utensils should be carried out as early as possible, helping reduce the residue of multispecies biofilms.
Other factors
In addition to the types of strains, EPS, and biofilm ages, there are other factors related to the disinfectant resistance of multispecies biofilms, such as environment and spatial distribution of strains. In terms of the environment, the surface shape can greatly affect the adhesion of microorganisms, especially when the surface has channels to protect the microorganisms from disinfectants. Depending on the physiological states of the bacteria and the molecules present during exposure, nutritional status can also alter the antibiotic sensitivity of multispecies biofilms (40). For example, the addition of CO2 could antagonize the effect of antibiotics on multispecies biofilms (39). Due to the specific spatial distribution of some species within the biofilms, some strains may be protected from disinfectants through aggregation with different three-dimensional structures from other strains (55). E. coli O157:H7 mainly appeared in the bottom layer of the dual-species biofilm, which provided R. insidiosa with better protection, including resistance to disinfection treatments (59). The cultivable local microorganisms from fresh salmon could form multispecies biofilms with L. monocytogenes, which had stronger antidisinfectant capacity than single-species biofilms, possibly because the honeycomb cell cluster shielded the underlying structures of the biofilms (80).
Overall, the enhanced resistance of multispecies biofilms to disinfectants may not depend on only one single factor but on a combination of multiple factors. For example, the biofilm age affects the production of EPS, which will, together, influence the disinfectant resistance of biofilms. Moreover, it is not feasible to predict the resistance of multispecies biofilms to disinfectants on the basis of the impact of a single species; this needs to be considered comprehensively according to the actual situation.
Interactions among microorganisms occur frequently and may eventually lead to the formation of dense, complex, and highly structured multispecies biofilms during food processing. The cooperative, competitive or neutral interactions between foodborne pathogens (such as Salmonella, L. monocytogenes, S. aureus, and E. coli) and other microorganisms in the biofilms may enhance pathogenicity. Understanding the preliminary mechanisms for the formation of multispecies biofilms can provide directions for blocking and eliminating the formation of some undesired biofilms. Using the antagonistic role of other microbial species may contribute to the development of methods for controlling pathogenic bacteria in food processing environments. However, the increased disinfectant resistance of most multispecies biofilms remains a great challenge in the food industry. In this article, the preliminary mechanisms for the resistance of multispecies biofilms to disinfectants were discussed, such as strain type, EPS, biofilm age, and nutritional status. Further exploration of these factors may help develop new and more efficient disinfectants. In addition, the combination of disinfectants with other methods (such as enzymes, bacteriophages, ultrasound, and ozone) may greatly improve the control efficiency on multispecies biofilms.
At present, the mechanism underlying the formation of multispecies biofilms is still not fully understood. Many laboratory-based studies of multispecies biofilms have been focused on low-diversity biofilms, such as biofilms consisting of two to four species. With the increase in cohabitating microorganisms, there will be a significant increase in the population number, which will increase the complexity of research. Although such studies may be difficult, it is a good way to explore the formation mechanisms of biofilms and the mechanisms for the disinfectant resistance mechanisms of multispecies biofilms with more than four species by using high-throughput sequencing techniques, such as transcriptomics, proteomics, and metabolomics. There are already many studies that have used confocal laser scanning microscopy and have three-dimensional renderings of multispecies biofilms. In the future, the dynamic process of multispecies biofilm formation can be explored by combining fluorescence in situ hybridization and confocal laser scanning microscopy and finally rendering three-dimensional visualization of the formation process of biofilms. Further studies of these issues will help find suitable and efficient ways to prevent and control the formation of multispecies biofilms in food processing environments.
This research was funded by Key Special Projects of Food Safety Key Technology R&D in 2019 (grant 2019YFC1605504).
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Russia : When and where to go?
The largest country in the world, Russia offers a climate with temperatures that get colder as you go from the South to the North and from the West to East as it turns into a more continental climate.
Winters are very cold; more so in Moscow than St. Petersburg with less snowfall in the latest. In spring, melting snow creates almost permanent mud everywhere in the country. At St. Petersburg, nights in June are clear as in all regions situated in the North because the sun doesn't really set. So, when planning to visit Russia, avoid the muddy spring and crowded July and August.
Autumn gives you the opportunity to admire a beautiful side of nature with its changing colours, and winter even though very cold has an undeniable charm: The snow is present but the cities are alive and vodka is flowing!
Historically rich, politically controversial, Russia is an amazing destination and not leave you unmoved.
Moscow of course is a must: cultural capital, it is also attractive for its medieval roots, and its sites become mythical, Red Square and the Kremlin, but also the Mayakovsky museum or zoo.
The other city where you can not ignore is St. Petersburg, which includes its canals and palaces, as at the time of the tsars, and offers of places like the legendary Peter and Paul Fortress and the Nevsky Prospekt.
At the far east, Vladivostok has recently opened to foreigners, and is the center of a nature reserve where there are tigers and bears and leopards.
Quite to the south, visit Sochi, a resort on the edge of the Black Sea, enjoying of a subtropical climate of warm waters that attract tourists from Russia and abroad, as well as spas and the surrounding hills dotted with dachas.
Not very far from Moscow, Novgorod dated 9th century teems with wonders, despite the devastation of the Nazis: the icons of the Palace of Facets, the historical museum, many churches, including that of the Transfiguration of the Savior with beautiful Byzantine frescoes.
Another curiosity not to be missed, the Trans-Siberian you will also travel from one end to another in this wonderful country for a distance of nearly a thousand miles for 6 days, making you go through huge forests of birch and pines and endless steppes, then along Lake Baikal and go to Irkutsk, a city very attractive and multicultural.
The Volga Volgograd, formerly Stalingrad, with its many museums and monuments of World War II, also trace a line of journey worthy of your attention to visit the center of the country.
Whatever your background, you'll be struck by the diversity of wealth in Russia, but also conquered by the famous Russian soul, responsible in turn of extreme joy and melancholy tenacity.
Moscow is connected to the principal cities of the world by daily flights, but getting there by train is also possible from Paris, Berlin or Helsinki and Prague.
To enter the country, you need a visa, you should also complete an immigration card at your arrival and register yourself at the 'OuFM'.
The currency in circulation is the ruble, it is divided into kopeks. It is relatively easy to withdraw money with a Visa or MasterCard at an ATM which are becoming easier to find.
Concerning your budget, without doubt the accommodation will be the most expensive part: In particular, accommodation in Moscow is very expensive; prices are often double compared to St. Petersburg. For a hotel, plan at least €70 for a double bedroom, often around €200 in Moscow. Another type of accommodation that is developing more and more is renting a renovated apartment which offers a good level of comfort: a studio can be found for around €60 the night, this is a good alternative to a hotel.
On the other hand, eating will definitely be less expensive (less than €15 for a meal) and you will have the choice between numerous specialties including various healthy and filling soups and salads.
Concerning sanitary precautions, it is recommended to have your usual vaccinations up to date (diphtheria, tetanus, polio) and to not drink tap water. Moreover, remember AIDS is very current in Russia: Do not have any sexual relations without protection.
To travel in the country, of course the train is your best choice, in particular the famous Trans Siberian that will take you up to Siberia, even Mongolia or Peking! Flying is also an interesting choice even if sometimes the comfort level is low for certain destinations. Renting a car is also a possible solution, both reasonable prices and road quality. In the principal cities, the underground is both practical and cheap.
Plan a budget for the numerous museums, very interesting but not cheap.


Most eukaryotic genes comprise exons and introns thus requiring the precise removal of introns from pre-mRNAs to enable protein biosynthesis. U2 and U12 spliceosomes catalyze this step by recognizing motifs on the transcript in order to remove the introns. A process which is dependent on precise definition of exon-intron borders by splice sites, which are consequently highly conserved across species. Only very few combinations of terminal dinucleotides are frequently observed at intron ends, dominated by the canonical GT-AG splice sites on the DNA level.
Here we investigate the occurrence of diverse combinations of dinucleotides at predicted splice sites. Analyzing 121 plant genome sequences based on their annotation revealed strong splice site conservation across species, annotation errors, and true biological divergence from canonical splice sites. The frequency of non-canonical splice sites clearly correlates with their divergence from canonical ones indicating either an accumulation of probably neutral mutations, or evolution towards canonical splice sites. Strong conservation across multiple species and non-random accumulation of substitutions in splice sites indicate a functional relevance of non-canonical splice sites. The average composition of splice sites across all investigated species is 98.7% for GT-AG, 1.2% for GC-AG, 0.06% for AT-AC, and 0.09% for minor non-canonical splice sites. RNA-Seq data sets of 35 species were incorporated to validate non-canonical splice site predictions through gaps in sequencing reads alignments and to demonstrate the expression of affected genes. We conclude that bona fide non-canonical splice sites are present and appear to be functionally relevant in most plant genomes, if at low abundance.


So you haven't yet upgraded to a phone with a reversible USB type-C cable? There is an app for that.
USB type-C Simulator is a mobile app somewhere in the category between a piece of art, social commentary, and a mobile game. The novelty app lets you insert and reverse a USB type-C cable to your hearts content in one of seven different virtual smart phones. That is actually all there is to the app; but it's quite well done for what it is. The game has a beautiful pixelated art style that compliments its simplicity quite well.
While the app concept and execution is very straight forward; players get treated to a fantastic sparkling rainbow display with fantastic accompanying music every time they insert the USB cable. The fabulous display that ensues from plugging in the cable should be enough to trigger the reward center in the primal part of just about anyone's brain.
Plug in for some joy!
The real joy of the game is to discover one of the new USB type-C cable connector's main benefits: there is no right-side-up! The cable works in any orientation!
You can unlock additional virtual phones in the game by repeatedly plugging and unplugging the cable, or by rotating the cable. Each task helps you towards completing quests which grant you larger bonus payouts.
The actual game isn't all that interesting. I was more fascinated by an app built around a cable connector standard. I doubt many non-technically minded people will ever download this app and learn about the wonders of the USB type-C connector. Although this app is something that Samsung or one of the other major phone vendors maybe should have created as a promotion for their own devices with type-C connectors. I believe this app could have been an effective and cheap advertisement for a specific device given a little PR help.
Update 2018 July : The app is no longer available on Google Play.
USB type C Simulator is available exclusively for Android on the Google Play Store. The app is cost-free but with advertisements.
Gameplay tip: You can tap the plug/unplug and reverse buttons at the same time to insert/remove the cable while rotating it. This strategy will let you maximize the bonuses money you can collect from completing quests. For any achievement hunters out there, there is an easy 87 000 points across 8 achievements in Google Play Games.
Please be aware that the app encourages you to repeatedly and rapidly tap on the same point on the screen only to throw up a full-screen advertisement that covers up the area you're clicking on. That is against the terms of service for most mobile advertisement networks, yet the app does it anyway.
This has only happened after you'e played the app for a little while, and then it happens a few seconds after your start the game. I do want to call out Adrian Zarzycki — the app's developer — for this obtrusive advertisement behavior and badly behaved app. Bad app developer. Bad.


Excellent Recreational and Timber tract. Property has ample dove, quail, duck and deer. The majority of the acreage is 25 year old planted pine stand and currently managed. There is a creek bed that offers mature hardwoods for wildlife.


text:
Booster Shots Provide Best Protection Against Omicron Variant, CDC Studies ShowThree new CDC studies from the CDC suggest a booster shot is the best protection against the Omicron variant.
This Seahawks Era Appears To Have Ended With A Russell Wilson Goal-Line Interception
Filed Under:Michael Hurley, New England Patriots, NFL, Pete Carroll, Russell Wilson, Seattle Seahawks, Sports News, Washington Football Team
By Michael Hurley, CBS Boston
BOSTON (CBS) — The "Legion Of Boom" broke up years ago, but the idea of the Seahawks as a perennial contender with Pete Carroll, Russell Wilson and Bobby Wagner remained intact — for the most part — over the last several seasons.
That illusion is now no more, after the Seahawks put forth one of the most miserable Monday night efforts possible in Washington, in a game that ended — appropriately — with a goal-line interception thrown by Russell Wilson.
It was, of course, a Russell Wilson goal-line interception that stopped the Seahawks from establishing a legitimate dynasty seven years ago, when the then-unknown Malcolm Butler came out of nowhere to pick off what would have been the Super Bowl-winning touchdown from Wilson.
Malcolm Butler's goal-line interception in Super Bowl XLIX.
Four years ago today. @Patriots @Mac_BZ pic.twitter.com/mP254qjQ7k
— NFL Throwback (@nflthrowback) February 1, 2019
Had the Seahawks done quite literally anything other than that, they would've won back-to-back Super Bowls, which hadn't been done by anybody in a decade and hasn't been done since. They would've had the makings of a dynasty, with a perfect combination of a nasty defense, a budding young superstar quarterback, an overwhelming running game, a head coach with the ideal personality for the locker room, and the best home-field advantage in the league.
With two straight Super Bowls in the bag — won against Peyton Manning and Tom Brady, no less — there would have been no telling how successful the Seahawks' run could have been.
Alas, Wilson's goal-line pick shattered that fantasy.
And since the Butler pick, the Seahawks have won just three playoff games. One was against the Lions, which is hardly an accomplishment. One came via a missed 27-yard field goal by Blair Walsh in Minnesota. The third came over a mediocre Eagles team that made the playoffs by default as the NFC East winner.
When facing actual playoff teams in the playoffs, the Seahawks lost to the Rams (2020), Packers (2019), Cowboys (2018), Falcons (2016), and Panthers (2015).
They won't get the chance to lose a playoff game this year, though, after they dropped to 3-8 with a moderately embarrassing 17-15 loss in Washington on Monday night.
After a three-and-out on their opening possession, Seattle went ona six-play, 75-yard touchdown drive to take a 7-3 lead late in the first quarter.
After that … here's how the next eight drives went for the Seahawks:
–Four plays, 22 yards, PUNT
–Two plays, 52 yards, FUMBLE
–Three plays, 9 yards, PUNT
–Three plays, -3 yards, PUNT
–Five plays, 16 yards, PUNT
Against one of the league's very worst passing defenses, that was just dreadful. Seattle's only points scored in a 44-minute stretch came on a blocked PAT returned for two points.
But Seattle did rise from their slumber for an exciting and successful drive in the final minutes of the game, with Wilson stepping up and finding a wide-open Freddie Swain for a touchdown with just seconds remaining on the clock.
Now trailing by two (because the Washington kicker coincidentally got injured while pursuing that blocked PAT return, thus forcing Washington to go for two after its own touchdown in the second half), the Seahawks obviously had to try a two-point conversion for the first time all season.
They opted to go with a pass. It ended poorly, but in familiar fashion for Wilson.
KENDALL FULLER.
INT on the two-point attempt! #SEAvsWAS pic.twitter.com/8WbyFDBVVy
— NFL (@NFL) November 30, 2021
In this instance, the interception didn't really matter. It doesn't count against Wilson in the stat sheet, as it came on a conversion attempt. An incompletion would have accomplished the same result for Washington.
But in terms of providing some good symmetry to the Seahawks' crumbling existence as contenders, a pick thrown by Wilson on a goal-line play is about as perfect as it gets.
(The Seahawks did recover the ensuing onside kick, but rather brutally they were penalized for an illegal formation, as one player who had nothing to do with the recovery lined up inside the opposite hash marks as opposed to outside the opposite hash marks. That's unfortunate, yes, as onside recoveries are incredibly rare these days. But it also reflects coaching.)
Carroll was a bit perplexed after the loss. He didn't know why Wilson's accuracy was so bad in the game, compared to practice. He didn't know why his kickoff team failed to line up properly, which cost his team one last-ditch shot at winning the game. He said Tyler Lockett might've been held on the failed two-point convesion. He had no explanation for D.K. Metcalf getting no looks for the bulk of the game.
The loss dropped the Seahawks to 3-8 on the season. They're in last place in the NFC West, and they have the second-worst record in the NFC, better only than the winless Lions. Aside from Detroit, only Houston and Jacksonville have worse records than the Seahawks in the entire NFL.
What's worse: The Seahawks don't even own their first-round pick in the 2022 draft. They traded that pick along with a first- and third-round pick in the 2021 draft, to the Jets in exchange for Jamal Adams. The idea behind that trade was likely that the addition of Adams could lift the Seahawks to an elite level. Instead, in a flash, they've faded into football obscurity.
The road to the bottom of the league was a long and winding one. But it began with a goal-line interception, and it was accentuated in the same fashion.
You can email Michael Hurley or find him on Twitter @michaelFhurley.

redpajama_set_name: RedPajamaCommonCrawl


On 12 July the Tent Academy Awards showcases the best Dutch and Finnish video and film art among the graduates of 2014. Video art is not the past; it is very much the future. New technologies make the world of video a very exciting place right now, which will be reflected in the TAA. During the award show on 12 July they will show the nominated clips, films, documentaries and animations, which will also be on view in the exhibition until 17 August. We talked to Joel Autio, one of the Finnish nominees, about the video 'Weep not for me, O mother'.
Fine, thanks! This year's been quite hectic, but I've had a chance to rest for a few days this week, hanging out with the dogs, taking naps.
How did you start making video art, and what do you like about it?
Cinema is a relatively young art form. There's still a lot to discover, to evolve, to transform. I believe it still has exceptional potential as an art, which I find very exciting. Since 2008 I've made a few short films and short documentaries, but the film you'll see at TAA is the first one I've made with a bigger crew, meaning more than 4 people were working on it. It was also the first project I chose to shoot on film instead of digital.
Can you tell me something about your video 'Weep not for me, O mother'? Is it scripted?
The name was taken from an Orthodox icon I saw nearly a year ago. The icon's name originates from 9th Ode of the Holy Saturday's Canon, in which the dead Christ comforts the mourning Saint Mary.
Nearly everything was scripted beforehand when we started to shoot the film last summer. There were more predefined restrictions than leaving room for improvisation: no camera movement, no focus pulling, every action should be shot without needing to cut it into pieces, etc. Despite all the restrictions I had created, I wanted the acting to feel as natural as possible. That was the biggest challenge for me as a director.
And, would you care to explain the narrative structure of the video? I noticed the hard cuts between the different scenes and the fact that you leave a lot open to interpretation, maybe you can say something about this.
There are a few seconds of darkness and silence between each scene. First, by using this method I wanted to break the feeling of continuity that would have occurred if I had cut all the scenes together. The film needs to be seen as a whole; a broken engine needs all of its parts to work. Second, I wanted to give the viewer a little time to think and feel what's happening on the screen; give a little time for the story to form into viewers mind, whatever it may be like.
Can you tell me about the 'feel' of the video, your use of film, the faded colours, the graininess, what are the considerations here?
When I was a child, once in a while we visited these family friends. Using a projector they showed us scratchy and grainy diafilm slides. In them there usually were just barren landscapes, old buildings and this eerie family. I found these slides rather boring. When looking at Weep not for me, O mother I like this reminiscence of watching a reversal film show, but with moving images. The empty landscapes and the strange family are still there, but this time they're alone, scattered and lost.
Obviously this nomination for TAA and the exhibition is pretty exciting, but do you have any other plans for the future, anything you're working on?
I'm writing a new short film script at the moment, which we're planning on shooting this summer with the same crew. This time there won't be restrictions.
The video 'Weep not for me, O Mother' by Joel Autio will be shown at the opening of Tent Academy Awards on 12 July, and will be on view in the exhibition at TENT until 17 August.


The CDC estimates there are approximately 2 million Americans living with autism and 1 in 68 children will be diagnosed with some degree of the disorder. Autism can be extremely difficult to cope with, especially for underprivileged families, that's why HALF the profits from ONEHOPE Cabernet help provide behavioral therapy for children with autism.
ONEHOPE donates HALF of profits generated from the sale of every bottle of Cabernet Sauvignon to benefit ACT Today! to help autistic children achieve their highest potential. The partnership betweenONEHOPE and ACT Today! helps provide funding and support for families who cannot afford specialized care for their autistic children. From social skills groups and referrals to biomedical treatments, ACT Today! is making positive change and providing hope to individuals with autism and their families.
Women in the U.S. have a 1 in 8 chance of developing breast cancer. ONEHOPE donates 50% of profits generated from the sale of every bottle of its Chardonnay to the National Breast Cancer Foundation. The National Breast Cancer Foundation is focused on education, prevention from early detection and treatment.
Trees provide us with everything from clean air and water, to climate health, to social and recreational benefits. Thanks to our partnership with American Forests, every bottle of Sauvignon Blanc plants a tree and helps to protect our forests and ensure these resources flourish for generations to come.
WINEMAKER ROB MONDAVI JR. - ONEHOPE Sauvignon Blanc is hand-crafted in partnership with Rob Mondavi, Jr. from select vineyards in Napa Valley, Sonoma County and California's Central Coast. Boasting a pale, golden color and flavors of pink grapefruit and bright, grassy undertones, this distinctive wine is fermented in stainless steel barrels. ONEHOPE Sauvignon Blanc is light, refreshing and finishes very crisp making it a perfect pairing with salmon, halibut, light pasta dishes, fresh fruits and a variety of cheeses.
ONEHOPE donates HALF of the profits from sales of its Sauvignon Blanc to benefit American Forests. For every bottle of Sauvignon Blanc, American Forests will plant a tree in a forest here in the US. American Forests encourages the long-term sustainability of America's forests, restoring wildlife habitat, and developing quality environmental education programs, to assure that Americans today, and in the future, enjoy healthy, growing forests.
Limited, only 5000 bottles for the U.S.
the brilliant 2012 Chateauneuf du Pape Chante Le Merle Vieilles Vignes is comprised of mostly old vine Grenache, with smaller parts Syrah and Mourvedre. Perfumed and already complex, with kirsch, blackberry, crushed stone, pepper and garrigue, this full-bodied effort has a silky, polished texture, big structure and serious length on the finish. It will mostly likely benefit for 3-4 years in the cellar and have 15-20 years of total longevity. Drink 2017-2032.
Looking just as good, with a slightly more approachable profile, the 2012 Chateauneuf du Pape A La Gloire de Mon Grand-Pere is a field blend of close to 100% Grenache that comes from the Gardiole lieu-dit. Aged all in demi-muid, it is an awesomely textured, seamless effort that boasts knockout aromas and flavors of kirsch, wild strawberries, herbs de Provence and red licorice. Not quite as backwards or concentrated as the Chante Le Merle, it should be approachable at an earlier age and drink brilliantly for 15 years or more. Drink 2015-2027.
From a vintage with miniscule yields and the same blend as the 2011, the 2012 Chateauneuf du Pape, which was tasted out of barrel, offers a pretty, fresh and fragrant profile with gorgeous purity of fruit. Black raspberry, licorice and floral notes all emerge here, and it builds in richness and depth with time in the glass. It should be reasonably approachable on release and drink well for 10-12 years. Drink 2014-2024.
Even better and showing plenty of similarities to the 2011, the 2012 Chateauneuf du Pape Vieilles Vignes is a 3,000-bottle lot of roughly 100% Grenache that was brought up all in stainless steel (this is due to not having enough juice to fill a larger barrel). Per Sophie, this cuvee was difficult to taste early on (which is a comment I heard often with regards to the 2012s) but had just recently turned the corner. Seamless, layered and textured, with overflowing Grenache generosity, this full-bodied effort gives up plenty of pure raspberry, black raspberry, violets and tinges of licorice on the nose and palate. As with the 2011, it should be reasonably approachable on release, yet have 15+ years or more of longevity. Drink 2015-2027.
Looking pretty close to the 2009 and 2010 in terms of quality, the 2012 Chateauneuf du Pape has slightly less Grenache than prior vintages, with 15% Mourvedre, 8% Syrah and the rest Grenache. Violets, underbrush, sweet black raspberry and cassis all flow from this medium to full-bodied, pure and elegantly styled effort. Showing plenty of fruit, good acidity and terrific purity, it should evolve gracefully for 12-15 years. Drink 2015-2027.
Also showing the purity and freshness of the vintage, the 2012 Chateauneuf du Pape Cuvee Chaupin, which was harvested towards the end of September, displays classic licorice, crushed violets, flowers and mineral-tinged black fruits to go with a medium to full-bodied, beautifully balanced and lively feel on the palate. Always 100% Grenache that comes from very old vines and sandy soils, it is one impressive tank sample. Give it a couple years in the cellar and enjoy bottles over the following decade or longer. Drink 2015-2027.
Most likely one of the top 2-3 wines produced, the 2012 Chateauneuf du Pape Vieilles Vignes is a rough blend of 80-85% Grenache, 10% Mourvedre (which was harvested October 10) and the balance Syrah, that hit 15.4% natural alcohol. Seeing the same elevage as the 2011, it sports an inky purple color as well as spectacular array of black and blue fruits, violets, liquid flowers and underbrush to go with an energetic, seamless and full-bodied profile on the palate. Lighter weight than the 2007 and 2010, it possesses notable freshness, purity and elegance. It should benefit from short-term cellaring and thrill for upward of two decades. Drink 2016-2032.
Still in tank, the 2012 Chateauneuf du Pape Tradition has plenty of floral, herb and underbrush notes to go with a core of sweet berry fruit. Medium to full-bodied, rich and textured, with ultra-fine tannin, it should be accessible in its youth yet have a solid 12-15 years drink window. Drink 2015-2027.
The distant drums of change are thumping, signaling the coming of chilling frosts, falling leaves and ghoulish screams. In a patch ripe with orange glow, Wilhelm Scream awakens and unleashes his season-stirring call for fall. His revelers listen for his echo across the ripened dancing days and prepare their seasonal celebration of harvest and ale.
Another unique creation from Mikkeller-Coffee IPA! The coffee is specially made by a coffee roaster called 'Koppi' in Sweden, and the "IPA" end is provided by generous amounts of Citra hops.
Saint Somewhere is a small microbrewery whose name was inspired by the Jimmy Buffet song, Boat Drinks (I gotta to fly to saint somewhere). They specialize in Belgian style ales and they only use Belgian malt.
Cannot find out much about this one.
In Mikkeller we have over the years done many collaboration brews. However, the last one we did was a little unusual – we collaborated with the Swedish tattoo studio Crooked Moon.
We have Cantillon in stock. Purchases up until Friday night at 7pm will count towards your BRC. I will email out Saturday morning.
4 Hands CA$H MONY will arrive tomorrow.
This one will be done via old school online reservations.
Bam Biere in half bottles!
Bam Bière - An artisan farmhouse ale that is golden, naturally cloudy, bottle conditioned and dry hopped for a perfectly refreshing balance of spicy malts, hops and yeast.
Mikkeller Spontan back in stock!
In the spontan series we present Spontan Gooseberry – this time, forming an alliance with gooseberry that were generously added underway the brewing process, creating a truly unique and distinct, sour beer.
In the spontan series we present Spontan Sea Buckthorn – this time, forming an alliance with Sea Buckthorn that were generously added underway the brewing process, creating a truly unique and distinct, sour beer.
In the spontan series we present Spontan Ellderflower – this time, forming an alliance with Elderflower that were generously added underway the brewing process, creating a truly unique and distinct, sour beer.
In the spontan series we present Spontan Mandarin – this time, forming an alliance with Mandarin that were generously added underway the brewing process, creating a truly unique and distinct, sour beer.
Called Beer Geek Bacon in Europe.
Mikkellers Beer Geek Bacon has now been released in a bottle edition. Smoke malt, coffee and hops makes this beer a unique experience.
As we compose the text for this celebratory beer‐a golden‐brown India pale ale dry-hopped exclusively and abundantly with El Dorado hops‐we stand ready to leap from the precipice of all we know into a new era for our company, our beers and, most importantly, our fans. It's one that will see us exploring new territory, both geographically and creatively, as we evolve from pacesetters for the American craft brewing movement to cross‐planetary champions of the worldwide cause of craft, hoisting our banner to proclaim artisanal beers' vast superiority over stripped‐down, business-driven, industrialized, fizzy, yellow mediocrity. We thank you for shadowing us for the first 18 years of our journey, and welcome you to join us for next leg and all that follows.
Lakefront Brewery's Pumpkin Lager is one of the only pumpkin lager available in the world; nearly all other pumpkin beers are ales. Using real pumpkin and a proprietary blend of spices made here in Milwaukee for us by the acclaimed Spice House, our brewers lager this beer for 4 full weeks. This extra time gives the Lakefront Pumpkin Lager a deep, smooth richness that no ale can match. A frothy entry leads to an off-dry medium-to-full body of intense cinnamon, nutmeg, cardamom, and candied yam flavor on a nutty wheat toast palate. Finishes with a drier spice and light toffee fade. Caramel and Munich malts reinforce this beer's mouthfeel and lend to the malty sweetness. A nicely balanced spice beer for those cool fall nights.
Out of Your Gourd Pumpkin Porter is dark chestnut brown in color and is made with pureed pumpkin. Cinnamon, nutmeg and ginger are added to the whirlpool and maple syrup is added during fermentation. This full-bodied, rich roasty porter makes you want to eat turkey and watch football, or build a bonfire.
Exciting new 12 pack from Sam Adams includes 2 bottles of each of these Single Hop IPAs.
Zeus hops contribute bitterness and an intense, pungent resinous pine flavor.
92 Point Anitnori at an Amazing price!


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When Terry joined the foreign service, I quit my job as an attorney in Philadelphia so that I could accompany him all over the world. I conveniently became pregnant two weeks after my last day at work and being a mom has been the bulk of my "employment" since then. Even when I was CLO, I was never away from home/the children more than 8 hours a day, 4 days a week - including commute.
What this all means is that the whole family is about to get thrown into a huge turmoil. Chores that have been mine for the last 7 years now need to be shared. My children won't even see me in the morning when they wake. I have a feeling I'll spend a lot less time on Facebook.
I am both excited to once again be a professional, and terrified about how the family will adjust to the new life.
On the whole, though, I am mostly excited - especially as each piece of the puzzle fits into place. Zoltan was easy - he can go full time to his preschool, and he's finally becoming comfortable there. Both days this week he played with everyone; he told me yesterday he likes speaking Russian.
For Alex there's been a lot more anxiety as those darned snow days last week made it impossible to call facilities to see if anyone had any space in March. As it turns out, the after school care center that I walked to in 10 minutes actually takes the kids outside EVERY DAY. In rain. In temps as low as 15F (yeah still plenty warm but lower than the school's outdoor recess policy so I'll take it gleefully). They tell parents to bring boots, umbrellas, whatever. They do kitchen projects like make vegan cookies or homemade play doh. Did I mention they go outside every day and are a 10 minute walk from home? They also have a summer camp that books up week by week so if there's any gaps in the Russian camp there's a backup.
The very last piece of the puzzle is morning care. I'm meeting a woman today and if it isn't a good vibe I have a few more applicants I can call. I am also amazingly lucky in the friends I have made in since we got here - two fellow moms have offered to pitch in if I end up short. Think about it. These people have known me barely 6 months and are willing to take my children in at 6:30am and get them to school. I didn't think this kind of thing happened outside the foreign service, where 6 months is an eternity and people become each other's "emergency contacts" within weeks.
Thanks for the tips Alex! I am super type A so the organization won't be too bad I think, just the execution :-) Oh and getting the rest of the family on board!
It's comforting to hear there will be quality time, I do have a weird fear of the kids forgetting who I am, or even worse, Zoltan not loving mommy best in the world anymore.


Rhytidectomy can improve the most visible signs of aging through the removal of excessive fat, repositioning of underlying muscles and removal of excess skin. A well-executed surgery has as principle to reposition the tissues in their original position, avoiding a "stretched" and artificial appearance. The various existing techniques, replete with individual details, are usually executed through incisions that start in the region of The temples, descend along the natural wrinkles in front of the ear and continue behind the ear to the scalp.
Anaesthesia may be general. However, some surgeons even use local anaesthesia under sedation, in special cases.
To perform the surgical procedure, both general and local anaesthesia with sedation are used. The final result will be the same. In terms of time, the surgery lasts about 3-6 hours for the face and front and 60 minutes for the eyelids.
It can vary between 12-36 hours, depending on the surgical technique used.
Until achieving the desired result, the patient evolves according to the phases characteristic of this type of surgery. They are edema (swelling), "spots" of blood infiltrates (ecchymosis), hypersensitivity of some areas, insensitivity of others. In general, they are common to all patients, varying according to individual characteristics. In most patients, after 2 weeks it is very difficult that the surgery has been performed.
Dry dressings are generally used. The large dressing is removed around the first 24 hours, leaving after that period, just a small dressing on the residual scars.
There is usually no pain in the postoperative period. The evolution of aesthetic surgeries is generally painless. Rarely, a case may present discreet malice, which can be perfectly evaluated with a common line analgesic.
Patients undergoing rhytidectomy should maintain absolute rest for 2 days, keeping the head always elevated for 7 days and avoiding physical exertion for approximately 3 weeks. Finally, try not to analyze the outcome of your surgery early. Remember that the swelling distorts the anatomical structures and has nothing to do with the final contour of the face! In most patients undergoing this surgery, this is hardly noticeable after 2-6 months.