Histological and Memory Alterations in an Innovative Alzheimer's Disease Animal Model by Vanadium Pentoxide Inhalation.

Background: Previous work from our group has shown that chronic exposure to Vanadium pentoxide (V2O5) causes cytoskeletal alterations suggesting that V2O5 can interact with cytoskeletal proteins through polymerization and tyrosine phosphatases inhibition, causing Alzheimer's disease (AD)-like hippocampal cell death. Objective: This work aims to characterize an innovative AD experimental model through chronic V2O5 inhalation, analyzing the spatial memory alterations and the presence of neurofibrillary tangles (NFTs), amyloid-ß (Aß) senile plaques, cerebral amyloid angiopathy, and dendritic spine loss in AD-related brain structures. Methods: 20 male Wistar rats were divided into control (deionized water) and experimental (0.02 M V2O5 1 h, 3/week for 6 months) groups (n = 10). The T-maze test was used to assess spatial memory once a month. After 6 months, histological alterations of the frontal and entorhinal cortices, CA1, subiculum, and amygdala were analyzed by performing Congo red, Bielschowsky, and Golgi impregnation. Results: Cognitive results in the T-maze showed memory impairment from the third month of V2O5 inhalation. We also noted NFTs, Aß plaque accumulation in the vascular endothelium and pyramidal neurons, dendritic spine, and neuronal loss in all the analyzed structures, CA1 being the most affected. Conclusions: This model characterizes neurodegenerative changes specific to AD. Our model is compatible with Braak AD stage IV, which represents a moment where it is feasible to propose therapies that have a positive impact on stopping neuronal damage.

Genome Engineering by RNA-Guided Transposition for Anabaena sp. PCC 7120.

In genome engineering, the integration of incoming DNA has been dependent on enzymes produced by dividing cells, which has been a bottleneck toward increasing DNA insertion frequencies and accuracy. Recently, RNA-guided transposition with CRISPR-associated transposase (CAST) was reported as highly effective and specific in Escherichia coli. Here, we developed Golden Gate vectors to test CAST in filamentous cyanobacteria and to show that it is effective in Anabaena sp. strain PCC 7120. The comparatively large plasmids containing CAST and the engineered transposon were successfully transferred into Anabaena via conjugation using either suicide or replicative plasmids. Single guide (sg) RNA encoding the leading but not the reverse complement strand of the target were effective with the protospacer-associated motif (PAM) sequence included in the sgRNA. In four out of six cases analyzed over two distinct target loci, the insertion site was exactly 63 bases after the PAM. CAST on a replicating plasmid was toxic, which could be used to cure the plasmid. In all six cases analyzed, only the transposon cargo defined by the sequence ranging from left and right elements was inserted at the target loci; therefore, RNA-guided transposition resulted from cut and paste. No endogenous transposons were remobilized by exposure to CAST enzymes. This work is foundational for genome editing by RNA-guided transposition in filamentous cyanobacteria, whether in culture or in complex communities.

Spatio-temporal coherence of circadian clocks and temporal control of differentiation in Anabaena filaments.

Circadian clock arrays in multicellular filaments of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 display remarkable spatio-temporal coherence under nitrogen-replete conditions. To shed light on the interplay between circadian clocks and the formation of developmental patterns, we followed the expression of a clock-controlled gene under nitrogen deprivation, at the level of individual cells. Our experiments showed that differentiation into heterocysts took place preferentially within a limited interval of the circadian clock cycle, that gene expression in different vegetative intervals along a developed filament was discoordinated, and that the circadian clock was active in individual heterocysts. Furthermore, Anabaena mutants lacking the kaiABC genes encoding the circadian clock core components produced heterocysts but failed in diazotrophy. Therefore, genes related to some aspect of nitrogen fixation, rather than early or mid-heterocyst differentiation genes, are likely affected by the absence of the clock. A bioinformatics analysis supports the notion that RpaA may play a role as master regulator of clock outputs in Anabaena, the temporal control of differentiation by the circadian clock and the involvement of the clock in proper diazotrophic growth. Together, these results suggest that under nitrogen-deficient conditions, the clock coherent unit in Anabaena is reduced from a full filament under nitrogen-rich conditions to the vegetative cell interval between heterocysts.IMPORTANCECircadian clocks, from unicellular organisms to animals, temporally align biological processes to day and night cycles. We study the dynamics of a circadian clock-controlled gene at the individual cell level in the multicellular filamentous cyanobacterium Anabaena, under nitrogen-stress conditions. Under these conditions, some cells along filaments differentiate to carry out atmospheric nitrogen fixation and lose their capability for oxygenic photosynthesis. We found that clock synchronization is limited to organismic units of contiguous photosynthetic cells, contrary to nitrogen-replete conditions in which clocks are synchronized over a whole filament. We provided evidence that the circadian clock regulates the process of differentiation, allowing it to occur preferentially within a limited time window during the circadian clock period. Lastly, we present evidence that the signal from the core clock to clock-regulated genes is conveyed in Anabaena as in unicellular cyanobacteria.

Evidence of validity and reliability of the environmental action scale in Peruvian university students.

Background: The environmental action scale is used to measure the degree of participation in collective environmental actions and has been shown to have adequate psychometric properties in developed countries. However, there are still no studies that have evaluated its performance in the Peruvian population. Methods: In this instrumental study, the environmental action scale (EAS) was translated, adapted, and validated. The EAS was administered to 352 university students between 18 and 35 years of age (Mage = 23.37, SD = 2.57) from different cities in Peru. A validity analysis was performed using two sources of evidence: content validity and internal structure, carrying out an exploratory factor analysis (EFA) and a confirmatory factor analysis (CFA). Results: The structure of the scale has been organized into three oblique factors. The findings confirmed the reliability and validity of the three dimensions of the EAS. Conclusion: Therefore, this scale is considered a valid option for assessing environmental action.

Heterologous expression of genes from a cyanobacterial endosymbiont highlights substrate exchanges with its diatom host.

A few genera of diatoms are widespread and thrive in low-nutrient waters of the open ocean due to their close association with N2-fixing, filamentous heterocyst-forming cyanobacteria. In one of these symbioses, the symbiont, Richelia euintracellularis, has penetrated the cell envelope of the host, Hemiaulus hauckii, and lives inside the host cytoplasm. How the partners interact, including how the symbiont sustains high rates of N2 fixation, is unstudied. Since R. euintracellularis has evaded isolation, heterologous expression of genes in model laboratory organisms was performed to identify the function of proteins from the endosymbiont. Gene complementation of a cyanobacterial invertase mutant and expression of the protein in Escherichia coli showed that R. euintracellularis HH01 possesses a neutral invertase that splits sucrose producing glucose and fructose. Several solute-binding proteins (SBPs) of ABC transporters encoded in the genome of R. euintracellularis HH01 were expressed in E. coli, and their substrates were characterized. The selected SBPs directly linked the host as the source of several substrates, e.g. sugars (sucrose and galactose), amino acids (glutamate and phenylalanine), and a polyamine (spermidine), to support the cyanobacterial symbiont. Finally, transcripts of genes encoding the invertase and SBPs were consistently detected in wild populations of H. hauckii collected from multiple stations and depths in the western tropical North Atlantic. Our results support the idea that the diatom host provides the endosymbiotic cyanobacterium with organic carbon to fuel N2 fixation. This knowledge is key to understanding the physiology of the globally significant H. hauckii-R. euintracellularis symbiosis.

Psychometric Network Analysis of the Problematic Pornography Use Scale (PPUS) in Peruvian Adults.

Objective: The aim of this research was to analyze the psychometric properties of the Problematic Pornography Use Scale (PPUS). Methods: A sample of 704 Peruvian youth and adults aged 18 to 62 years (M = 26, SD = 6.0) was considered, of which 56% were female and 43% male. The participants were from various cities in Peru: Lima (84%), Trujillo (2.6%), Arequipa (1.8%), and Huancayo (1.6%). The validity of the theoretical structure of the PPUS was carried out by means of two techniques: Confirmatory factor analysis (CFA) and Exploratory Graphical Analysis (EGA), a new effective and efficient dimension evaluation technique, whose measure consists of verifying the fit of the dimension structure. Results: Using the bifactor model, the hypothesis that PPUS has a unifactorial behavior was corroborated. These approximations of unidimensionality are also corroborated by the EGA method, where the centrality parameters and network loadings are found to have acceptable estimates. Conclusion: The results demonstrate the validity of the PPUS, contrasting with the factor model and verifying the unidimensionality of the construct, which provide useful directions for future studies on the instrumentalization of problematic pornography use scale.

Intervenciones quirúrgicas mínimamente invasivas complementarias a la ortodoncia quirúrgicamente asistida: Resumen estructurado de evidencia

Introducción: Existen procedimientos quirúrgicos que pueden generar una disminución en la duración de los tratamientos de ortodoncia (TO) mediante una aceleración del movimiento dental. La técnica más estudiada corresponde a la corticotomía clásica, la cual muchas veces es desechada por los pacientes debido a su invasividad. Es por esto que nacen las intervenciones quirúrgicas mínimamente invasivas (IQMI), tales como las micro osteoperforaciones (MOP) y la piezocisión, que buscan el mismo resultado, pero sin realizar colgajos de espesor total, otorgándole al paciente nuevas alternativas terapéuticas para acortar el tratamiento de ortodoncia. La evidencia al respecto aún es controversial, debido a que la certeza de la evidencia es baja o muy baja con relación a estos procedimientos. Métodos: Realizamos una búsqueda en Epistemonikos, la mayor base de datos de revisiones sistemáticas en salud, la cual es mantenida mediante el cribado de múltiples fuentes de información, incluyendo MEDLINE, EMBASE, Cochrane, entre otras. Extrajimos los datos desde las revisiones identificadas, analizamos los datos de los estudios primarios, realizamos un metaanálisis y preparamos una tabla de resumen de los resultados utilizando el método GRADE. Resultados y conclusiones: Identificamos 39 revisiones sistemáticas que en conjunto incluyeron 43 estudios primarios, de los cuales, 31 corresponden a ensayos aleatorizados. Concluimos que las intervenciones quirúrgicas mínimamente invasivas podrían aumentar la tasa de movimiento dental a las 12 semanas, la distancia total acumulada, la tasa de movimiento dental y reducir la duración total de tratamiento, pero la certeza de la evidencia es incierta. Además, podrían resultar en poca o ninguna diferencia sobre el índice gingival, la profundidad de sondaje y el índice de placa.

Introduction: There are surgical procedures that can generate a decrease in the orthodontic (OT) treatments duration through a Acceleration of tooth movement. The most studied technique corresponds to classical corticotomy, which is often discarded by patients due to its invasiveness. This is why minimally invasive surgical interventions (MISI) are born, such as micro osteoperforations (MOP) and piezocision, which seek the same result, but without making total thickness flaps, giving the patient new therapeutic alternatives to shorten orthodontic treatment. The evidence on this is still controversial, because the certainty of the evidence is low or very low in relation to these procedures. Methods: A search was performed using Epistemonikos, the biggest database for systematic reviews in health, which is maintained by screening of multiple sources of information, including MEDLINE, EMBASE, Cochrane, among others. Data from systematic reviews were extracted, and analysis of the primary studies was performed, including a meta-analysis and a summary of findings table using GRADE approach. Results and conclusions: We identified 39 systematic reviews that together included 43 primary studies, of which 31 correspond to randomized clinical trials. We conclude that minimally invasive surgical interventions could increase the rate of tooth movement at 12 weeks, distance total accumulated, the rate of tooth movement and reduce the total duration of treatment, but the certainty of the evidence is uncertain. In addition, they could result in little or no difference in gingival index, probing depth and plaque index.

Microplastic abundance in feces of lagomorphs in relation to urbanization.

The presence of microplastics (MPs) in marine environments has been extensively documented. However, studies of terrestrial species are scarce. Fecal samples (105) of lagomorphs were collected at sites with different levels of urbanization in the Baja California Chaparral and analyzed to quantify and characterize MPs found in the feces. The lagomorph species recorded in the study area are the desert cottontail rabbit (Sylvilagus audubonii), brush rabbit (Sylvilagus bachmani), and black-tailed jackrabbit (Lepus californicus), which play important roles in the food web of the chaparral ecosystem. Microplastics were identified using attenuated total reflectance Fourier transform infrared spectroscopy. Microplastics were detected in 49 % of the samples, with fibers being the dominant shape found (72 %). Most (75 %) of the MPs were <1 mm in size, with a mean length of 0.93 ± 0.99 mm (median 0.60 mm, range 0.02 - <5 mm). Polyamide was the dominant polymer (54 %), indicating that MPs are likely derived from textiles; polyethylene was also abundant (27 %). A difference was also observed in the abundance of MPs in feces from sites with different levels of urbanization, with the highest abundance in feces from the urban sites.

Post-cardioversion time Course of Atrial Remodeling Markers and their Association with Recurrence in Subjects with Long-standing, Persistent Atrial Fibrillation.

BACKGROUND: Activation of the renin-angiotensin-aldosterone axis with elevation of inflammatory markers and the resulting fibrosis play a very important role in atrial remodeling in patients with atrial fibrillation (AF), which is associated with post-cardioversion recurrence. AIM OF THE STUDY: The purpose of the study was to describe the time course of angiotensin II (AngII), aldosterone, and of the amino terminal pro-peptide of type III pro-collagen (PIIINP) following cardioversion, and their association with arrhythmia recurrence. METHODS: Ninety-nine subjects with long-standing, persistent, non-valvular atrial fibrillation who underwent successful electrical cardioversion were included, with a 6 month follow up. Angiotensin II (AngII), aldosterone and PIIINP concentrations were measured at 0, 1, 7, 30, and 180 d. Two groups were formed for the analysis: continuing sinus rhythm and recurrence of AF. RESULTS: 53% of the subjects experienced recurrence of AF. Subjects with recurrence had larger left atrial diameters and lower global peak atrial longitudinal strain (8.7 vs. 19.7%; p <0.001), higher levels of AngII (431.85 vs. 257.97 pg/mL; p = 0.003) at 180 d, higher pre-cardioversion levels of aldosterone, (11.42 vs. 5.46 pg/mL; p = 0.048) at 1 d (12.01 vs. 5.05 pg/mL; p = 0.004) and at 180 d (12.66 vs. 7.51 pg/mL; p = 0.011). There were no differences in PIIINP levels between both groups. CONCLUSIONS: Electrical post-cardioversion recurrence in subjects with long-standing, persistent AF is associated with elevated levels of AngII and aldosterone.

Systematic mapping review of orthognathic surgery.

STUDY DESIGN: Systematic mapping review AIM AND SCOPE: The objective of this mapping review was to identify, describe, and organize clinical research currently available from systematic reviews and primary studies regarding co-interventions and different surgical modalities used in orthognathic surgery (OS) and their outcomes. METHODS: Systematic reviews (SRs), randomized controlled trials, and observational studies that evaluated perioperative OS co-interventions and surgical modalities were identified in an exhaustive search of MEDLINE, EMBASE, Epistemonikos, Lilacs, Web of Science, and CENTRAL. Grey literature was also screened. RESULTS: Included were 35 SRs and 253 primary studies, 103 from SRs, and another 150 identified in our search. Overall, SR quality was rated as critically low, with only two SRs rated as of high quality. 19 questions on population, interventions, comparisons, and outcomes (PICO) extracted from the SRs focused on osteosynthesis methods, surgical cutting devices, and use of antibiotics, corticosteroids, and induced hypotension. Also identified were 15 research gaps. Evidence bubble maps were created to graphically depict the available evidence. CONCLUSION: Future high-quality research, both primary and secondary, is needed to address the knowledge gaps identified in this systematic mapping review.

Adaptation to an Intracellular Lifestyle by a Nitrogen-Fixing, Heterocyst-Forming Cyanobacterial Endosymbiont of a Diatom.

The symbiosis between the diatom Hemiaulus hauckii and the heterocyst-forming cyanobacterium Richelia intracellularis makes an important contribution to new production in the world's oceans, but its study is limited by short-term survival in the laboratory. In this symbiosis, R. intracellularis fixes atmospheric dinitrogen in the heterocyst and provides H. hauckii with fixed nitrogen. Here, we conducted an electron microscopy study of H. hauckii and found that the filaments of the R. intracellularis symbiont, typically composed of one terminal heterocyst and three or four vegetative cells, are located in the diatom's cytoplasm not enclosed by a host membrane. A second prokaryotic cell was also detected in the cytoplasm of H. hauckii, but observations were infrequent. The heterocysts of R. intracellularis differ from those of free-living heterocyst-forming cyanobacteria in that the specific components of the heterocyst envelope seem to be located in the periplasmic space instead of outside the outer membrane. This specialized arrangement of the heterocyst envelope and a possible association of the cyanobacterium with oxygen-respiring mitochondria may be important for protection of the nitrogen-fixing enzyme, nitrogenase, from photosynthetically produced oxygen. The cell envelope of the vegetative cells of R. intracellularis contained numerous membrane vesicles that resemble the outer-inner membrane vesicles of Gram-negative bacteria. These vesicles can export cytoplasmic material from the bacterial cell and, therefore, may represent a vehicle for transfer of fixed nitrogen from R. intracellularis to the diatom's cytoplasm. The specific morphological features of R. intracellularis described here, together with its known streamlined genome, likely represent specific adaptations of this cyanobacterium to an intracellular lifestyle.

Genetic and lipidomic analyses suggest that Nostoc punctiforme, a plant-symbiotic cyanobacterium, does not produce sphingolipids.

Sphingolipids, a class of amino-alcohol-based lipids, are well characterized in eukaryotes and in some anaerobic bacteria. However, the only sphingolipids so far identified in cyanobacteria are two ceramides (i.e., an acetylsphingomyelin and a cerebroside), both based on unbranched, long-chain base (LCB) sphingolipids in Scytonema julianum and Moorea producens , respectively. The first step in de novo sphingolipid biosynthesis is the condensation of l-serine with palmitoyl-CoA to produce 3-keto-diyhydrosphingosine (KDS). This reaction is catalyzed by serine palmitoyltransferase (SPT), which belongs to a small family of pyridoxal phosphate-dependent α-oxoamine synthase (AOS) enzymes. Based on sequence similarity to molecularly characterized bacterial SPT peptides, we identified a putative SPT (Npun_R3567) from the model nitrogen-fixing, plant-symbiotic cyanobacterium, Nostoc punctiforme strain PCC 73102 (ATCC 29133). Gene expression analysis revealed that Npun_R3567 is induced during late-stage diazotrophic growth in N. punctiforme . However, Npun_R3567 could not produce the SPT reaction product, 3-keto-diyhydrosphingosine (KDS), when heterologously expressed in Escherichia coli . This agreed with a sphingolipidomic analysis of N. punctiforme cells, which revealed that no LCBs or ceramides were present. To gain a better understanding of Npun_R3567, we inferred the phylogenetic position of Npun_R3567 relative to other bacterial AOS peptides. Rather than clustering with other bacterial SPTs, Npun_R3567 and the other cyanobacterial BioF homologues formed a separate, monophyletic group. Given that N. punctiforme does not appear to possess any other gene encoding an AOS enzyme, it is altogether unlikely that N. punctiforme is capable of synthesizing sphingolipids. In the context of cross-kingdom symbiosis signalling in which sphingolipids are emerging as important regulators, it appears unlikely that sphingolipids from N. punctiforme play a regulatory role during its symbiotic association with plants.

Functional Diversity of TonB-Like Proteins in the Heterocyst-Forming Cyanobacterium Anabaena sp. PCC 7120.

The TonB-dependent transport of scarcely available substrates across the outer membrane is a conserved feature in Gram-negative bacteria. The plasma membrane-embedded TonB-ExbB-ExbD accomplishes complex functions as an energy transducer by physically interacting with TonB-dependent outer membrane transporters (TBDTs). TonB mediates structural rearrangements in the substrate-loaded TBDTs that are required for substrate translocation into the periplasm. In the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, four TonB-like proteins have been identified. Out of these TonB3 accomplishes the transport of ferric schizokinen, the siderophore which is secreted by Anabaena to scavenge iron. In contrast, TonB1 (SjdR) is exceptionally short and not involved in schizokinen transport. The proposed function of SjdR in peptidoglycan structuring eliminates the protein from the list of TonB proteins in Anabaena. Compared with the well-characterized properties of SjdR and TonB3, the functions of TonB2 and TonB4 are yet unknown. Here, we examined tonB2 and tonB4 mutants for siderophore transport capacities and other specific phenotypic features. Both mutants were not or only slightly affected in schizokinen transport, whereas they showed decreased nitrogenase activity in apparently normal heterocysts. Moreover, the cellular metal concentrations and pigment contents were altered in the mutants, most pronouncedly in the tonB2 mutant. This strain showed an altered susceptibility toward antibiotics and SDS and formed cell aggregates when grown in liquid culture, a phenotype associated with an elevated lipopolysaccharide (LPS) production. Thus, the TonB-like proteins in Anabaena appear to take over distinct functions, and the mutation of TonB2 strongly influences outer membrane integrity. IMPORTANCE The genomes of many organisms encode more than one TonB protein, and their number does not necessarily correlate with that of TonB-dependent outer membrane transporters. Consequently, specific as well as redundant functions of the different TonB proteins have been identified. In addition to a role in uptake of scarcely available nutrients, including iron complexes, TonB proteins are related to virulence, flagellum assembly, pilus localization, or envelope integrity, including antibiotic resistance. The knowledge about the function of TonB proteins in cyanobacteria is limited. Here, we compare the four TonB proteins of Anabaena sp. strain PCC 7120, providing evidence that their functions are in part distinct, since mutants of these proteins exhibit specific features but also show some common impairments.

Alzheimer-like cell death after vanadium pentoxide inhalation.

Vanadium (V) toxicity depends on its oxidation state; it seems that vanadium pentoxide (V2O5) is the most toxic to the living cells. It has been reported that oral administration induces changes in motor activity and learning; in rats, I.P. administration increases lipid peroxidation levels in the cerebellum and the concentration of free radicals in the hippocampus and cerebellum. Mice that inhaled V2O5 presented a reduced number of tubulin+ in Leydig and Sertoli cells; it has also been reported that inhaled V2O5 induces loss of dendritic spines, necrosis, and hippocampus neuropil alterations; considering the direct consequence of the interaction of V with cytoskeletal components, makes us believe that V2O5 exposure could cause neuronal death in the hippocampus similar to that seen in Alzheimer disease. This work aimed to determine pyramidal hippocampal CA1 cytoskeletal alterations with Bielschowsky stain in rats exposed to V2O5. Male Wistar rats inhaled 0.02 M of V2O5 one h two times a week for two and six months. We found that rats, which inhaled V2O5 reached 56,57% of dead neurons after six months of inhalation; we recognize strong argyrophilic and collapsed somas and typical flame-shaped in all V-exposed rats hippocampus CA1 compared to controls. We also observe somatodendritic distortions. Axons and dendrites displayed thick dark bands replaced by noticeable thickening and nodosities and the cytoskeleton fibrillary proteins' linear traces. Our findings suggest that V2O5 inhalation induces Alzheimer-like cell death with evident cytoskeletal alterations.

Single-Cell Measurements of Fixation and Intercellular Exchange of C and N in the Filaments of the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120.

Under diazotrophic conditions, the model filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 develops a metabolic strategy based on the physical separation of the processes of oxygenic photosynthesis, in vegetative cells, and N2 fixation, in heterocysts. This strategy requires the exchange of carbon and nitrogen metabolites and their distribution along the filaments, which takes place through molecular diffusion via septal junctions involving FraCD proteins. Here, Anabaena was incubated in a time course (up to 20 h) with [13C]bicarbonate and 15N2 and analyzed by secondary ion mass spectrometry imaging (SIMS) (large-geometry SIMS [LG-SIMS] and NanoSIMS) to quantify C and N assimilation and distribution in the filaments. The 13C/12C and 15N/14N ratios measured in wild-type filaments showed a general increase with time. The enrichment was relatively homogeneous in vegetative cells along individual filaments, while it was reduced in heterocysts. Heterocysts, however, accumulated recently fixed N at their poles, in which the cyanophycin plug [multi-l-arginyl-poly(l-aspartic acid)] is located. In contrast to the rather homogeneous label found along stretches of vegetative cells, 13C/12C and 15N/14N ratios were significantly different between filaments both at the same and different time points, showing high variability in metabolic states. A fraC fraD mutant did not fix N2, and the 13C/12C ratio was homogeneous along the filament, including the heterocyst in contrast to the wild type. Our results show the consumption of reduced C in the heterocysts associated with the fixation and export of fixed N and present an unpredicted heterogeneity of cellular metabolic activity in different filaments of an Anabaena culture under controlled conditions. IMPORTANCE Filamentous, heterocyst-forming cyanobacteria represent a paradigm of multicellularity in the prokaryotic world. Physiological studies at the cellular level in model organisms are crucial to understand metabolic activities and qualify specific aspects related to multicellularity. Here, we used stable isotopes (13C and 15N) coupled to LG-SIMS and NanoSIMS imaging to follow single-cell C and N2 fixation and metabolic dynamics along the filaments in the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. Our results show a close relationship between C and N fixation and distribution in the filaments and indicate that wild-type filaments in a culture can exhibit a substantial variability of metabolic states. This illustrates how some novel properties can be appreciated by studying microbial cultures at the single-cell level.

A TonB-Like Protein, SjdR, Is Involved in the Structural Definition of the Intercellular Septa in the Heterocyst-Forming Cyanobacterium Anabaena.

Cyanobacteria are photosynthetic organisms with a Gram-negative envelope structure. Certain filamentous species such as Anabaena sp. strain PCC 7120 can fix dinitrogen upon depletion of combined nitrogen. Because the nitrogen-fixing enzyme, nitrogenase, is oxygen sensitive, photosynthesis and nitrogen fixation are spatially separated in Anabaena. Nitrogen fixation takes place in specialized cells called heterocysts, which differentiate from vegetative cells. During heterocyst differentiation, a microoxic environment is created by dismantling photosystem II and restructuring the cell wall. Moreover, solute exchange between the different cell types is regulated to limit oxygen influx into the heterocyst. The septal zone containing nanopores for solute exchange is constricted between heterocysts and vegetative cells, and cyanophycin plugs are located at the heterocyst poles. We identified a protein previously annotated as TonB1 that is largely conserved among cyanobacteria. A mutant of the encoding gene formed heterocysts but was impaired in diazotrophic growth. Mutant heterocysts appeared elongated and exhibited abnormal morphological features, including a reduced cyanophycin plug, an enhanced septum size, and a restricted nanopore zone in the septum. In spite of this, the intercellular transfer velocity of the fluorescent marker calcein was increased in the mutant compared to the wild type. Thus, the protein is required for proper formation of septal structures, expanding our emerging understanding of Anabaena peptidoglycan plasticity and intercellular solute exchange, and is therefore renamed SjdR (septal junction disk regulator). Notably, calcium supplementation compensated for the impaired diazotrophic growth and alterations in septal peptidoglycan in the sjdR mutant, emphasizing the importance of calcium for cell wall structure. IMPORTANCE Multicellularity in bacteria confers an improved adaptive capacity to environmental conditions and stresses. This includes an enhanced capability of resource utilization through a distribution of biochemical processes between constituent cells. This specialization results in a mutual dependency of different cell types, as is the case for nitrogen-fixing heterocysts and photosynthetically active vegetative cells in Anabaena. In this cyanobacterium, intercellular solute exchange is facilitated through nanopores in the peptidoglycan between adjacent cells. To ensure functionality of the specialized cells, septal size as well as the position, size, and frequency of nanopores in the septum need to be tightly established. The novel septal junction disk regulator SjdR characterized here is conserved in the cyanobacterial phylum. It influences septal size and septal nanopore distribution. Consequently, its absence severely affects the intercellular communication and the strains' growth capacity under nitrogen depletion. Thus, SjdR is involved in septal structure remodeling in cyanobacteria.

Heterocyst Septa Contain Large Nanopores That Are Influenced by the Fra Proteins in the Filamentous Cyanobacterium Anabaena sp. Strain PCC 7120.

Multicellular heterocyst-forming cyanobacteria, such as Anabaena, grow as chains of cells forming filaments that, under diazotrophic conditions, contain two cell types: vegetative cells that perform oxygenic photosynthesis and N2-fixing heterocysts. Along the filament, the intercellular septa contain a thick peptidoglycan layer that forms septal disks. Proteinaceous septal junctions connect the cells in the filament traversing the septal disks through nanopores. The fraCDE operon encodes proteins needed to make long filaments in Anabaena. FraC and FraD, located at the intercellular septa, are involved in the formation of septal junctions. Using a superfolder-green fluorescent protein (GFP) fusion, we found in this study that FraE is mainly localized to the poles of the heterocysts, consistent with the requirement of FraE for constriction of the heterocyst poles to form the "heterocyst neck." A fraE insertional mutant was impaired by 22% to 38% in transfer of fluorescent calcein from vegetative cells to heterocysts. Septal disks were inspected in murein sacculi from heterocyst-enriched preparations. Unexpectedly, the diameter of the nanopores in heterocyst septa was about 1.5- to 2-fold larger than in vegetative cell septa. The number of these nanopores was 76% and 6% of the wild-type number in fraE and fraC fraD mutants, respectively. Our results show that FraE is mainly involved in heterocyst maturation, whereas FraC and FraD are needed for the formation of the large nanopores of heterocyst septa, as they are for vegetative cell nanopores. Additionally, arrays of small pores conceivably involved in polysaccharide export were observed close to the septal disks in the heterocyst murein sacculus preparations. IMPORTANCE Intercellular communication, an essential attribute of multicellularity, is required for diazotrophic growth in heterocyst-forming cyanobacteria such as Anabaena, in which the cells are connected by proteinaceous septal junctions that are structural analogs of metazoan connexons. The septal junctions allow molecular intercellular diffusion traversing the septal peptidoglycan through nanopores. In Anabaena the fraCDE operon encodes septal proteins involved in intercellular communication. FraC and FraD are components of the septal junctions along the filament, whereas here we show that FraE is mainly present at the heterocyst poles. We found that the intercellular septa in murein sacculi from heterocysts contain nanopores that are larger than those in vegetative cells, establishing a previously unknown difference between heterocyst and vegetative cell septa in Anabaena.