The lolB gene in Xanthomonas campestris pv. campestris is required for bacterial attachment, stress tolerance, and virulence

Background Xanthomonas campestris pv. campestris (Xcc) is a Gram-negative bacterium that can cause black rot disease in crucifers. The lipoprotein outer membrane localization (Lol) system is involved in the lipoprotein sorting to the outer membrane. Although Xcc has a set of annotated lol genes, there is still little known about the physiological role in this phytopathogen. In this study, we aimed to characterize the role of LolB of Xcc in bacterial attachment, stress tolerance, and virulence. Results To characterize the role of LolB, lolB mutant was constructed and phenotypic evaluation was performed. The lolB mutant revealed reductions in bacterial attachment, extracellular enzyme production, and virulence. Mutation of lolB also resulted in reduced tolerance to a myriad of stresses, including heat and a range of membrane-perturbing agents. Trans-complementation of lolB mutant with intact lolB gene reverted these altered phenotypes to the wild-type levels. From subsequent reporter assay and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis, the expression of genes that encode the major extracellular enzymes and the stress-related proteins was reduced after lolB mutation. Conclusions The results in this work contribute to the functional understanding of lolB in Xanthomonas for the first time, and provide new insights into the function of lolB in bacteria.

Gene expression in the amygdala and hippocampus of cyclic and acyclic gilts

Age at first estrus is the earliest phenotypic indicator of future reproductive success of gilts. Prebreeding anestrus is a major reason for reproductive failure leading to culling of replacement gilts. The two types of prebreeding anestrus are delay in attaining puberty (prepubertal anestrus, PPA) and silent ovulation (behavioral anestrus, BA). Neural tissues such as amygdala and hippocampus play a major role in regulating sexual behavior, social interactions, and receptivity to males. Differences in gene expression in the amygdala and hippocampus of gilts were analyzed in three comparisons; 1) PPA cases and cyclic controls at follicular phase of estrous cycle, 2) BA cases and cyclic controls at luteal phase of estrous cycle, and 3) gilts at different stages of the ovarian cycle (cyclic gilts at follicular phase and luteal phase of estrous cycle) to gain functional understanding of how these rarely studied tissues may differ between pubertal phenotypes and different stages of the estrous cycle of gilts. Differentially expressed genes (DEG) between PPA and BA cases and their respective cyclic controls were involved in neurological and behavioral disorders as well as nervous system functions that could directly or indirectly involved in development of behaviors related to estrus. The comparison between cyclic follicular and luteal phase control gilts identified the greatest number of DEG in the hippocampus and amygdala. These DEG were involved in adult neurogenesis and neural synapse (e.g., GABAergic, dopamine, cholinergic) suggesting that these tissues undergo structural changes and synaptic plasticity in gilts. This is the first report to demonstrate that the stage of estrous cycle is associated with dynamic changes in gene expression within porcine hippocampus and amygdala and indicates a role of gonadal steroids in regulating their biology.

Computed tomography examination of the os cordis in a lamb (Ovis aries Linnaeus, 1758)

Objective. The present paper aims to investigate the role of computed tomography as an imaging technique of diagnosis in the identification of the os cordis in ovine, and also how this anatomical structure is morphotopographically characterized in macro and microscopic contexts, seeking to contribute for its functional understanding. Materials and method. The heart of a young male ovine had been donated to the Laboratory of Animal Anatomy of the Surgery Department of the FMVZ/USP, first being subject to a post-mortem examination by means of the cardiac “shedding” (transverse cross sectioning of the heart). A tomographic examination of the anatomic specimen was carried out, as well as the dissection and histological analysis of the collected sample. Results. The results indicate the presence of an osseous structure of 6.39 mm in length, located in the aortic valve of the heart, next to a ring comprised by three valve flaps, tendinous cords, and papillary muscles. The histological findings consist of fibrous connective tissue, cancellous bone tissue, and calcified hyaline cartilage wherein the cardiomyocytes are attached. Conclusions. It is concluded that computed tomography, even though seldom applied to animals of zootechnical interest, poses as an effective tool for the visualization of the os cordis in lambs.

Proteome of the Luminal Surface of the Blood–Brain Barrier

Interrogation of the molecular makeup of the blood–brain barrier (BBB) using proteomic techniques has contributed to the cataloguing and functional understanding of the proteins uniquely organized at this specialized interface. The majority of proteomic studies have focused on cellular components of the BBB, including cultured brain endothelial cells (BEC). Detailed proteome mapping of polarized BEC membranes and their intracellular endosomal compartments has led to an improved understanding of the processes leading to internalization and transport of various classes of molecules across the BBB. Quantitative proteomic methods have further enabled absolute and comparative quantification of key BBB transporters and receptors in isolated BEC and microvessels from various species. However, translational studies further require in vivo/in situ analyses of the proteins exposed on the luminal surface of BEC in vessels under various disease and treatment conditions. In vivo proteomics approaches, both profiling and quantitative, usually rely on ‘capturing’ luminally-exposed proteins after perfusion with chemical labeling reagents, followed by analysis with various mass spectrometry-based approaches. This manuscript reviews recent advances in proteomic analyses of luminal membranes of BEC in vitro and in vivo and their applications in translational studies focused on developing novel delivery methods across the BBB.

A spatial fingerprint of land-water linkage of biodiversity uncovered by remote sensing and environmental DNA

Aquatic and terrestrial ecosystems are tightly connected via spatial flows of organisms and resources. Such land-water linkages integrate biodiversity across ecosystems and suggest a spatial association of aquatic and terrestrial biodiversity. However, knowledge about this spatial extent is limited. By combining satellite remote sensing (RS) and environmental DNA (eDNA) extraction from river water across a 740-km2 mountainous catchment, we identify a characteristic spatial land-water fingerprint. Specifically, we find a spatial association of riverine eDNA diversity with RS spectral diversity of terrestrial ecosystems upstream, peaking at a 400 m distance yet still detectable up to a 3.3 km radius. Our findings testify that biodiversity patterns in rivers can be linked to the functional diversity of surrounding terrestrial ecosystems and provide a dominant scale at which these linkages are strongest. Such spatially explicit information is necessary for a functional understanding of land-water linkages and provides a reference scale for adequate conservation and landscape management decisions.

α-/γ-Taxilin are required for centriolar subdistal appendage assembly and microtubule organization

The centrosome, composed of a pair of centrioles (mother and daughter centrioles) and pericentriolar material, is mainly responsible for microtubule nucleation and anchorage in animal cells. The subdistal appendage (SDA) is a centriolar structure located at the subdistal region on the mother centriole, and it functions in microtubule anchorage. However, the molecular composition and detailed structure of SDA remain largely unknown. Here, we identified a-taxilin and r-taxilin as new SDA components, which form a complex via their coiled-coil domains and serve as a new subgroup during SDA hierarchical assembly. Their SDA localization is dependent on ODF2, and α-taxilin recruits CEP170 to the SDA. Functional analyses suggest that α-taxilin and γ-taxilin are responsible for centrosomal microtubule anchorage during interphase, as well as for proper spindle orientation during metaphase. Altogether, our results shed light on the molecular components and functional understanding of the SDA hierarchical assembly and microtubule organization.

Processive dynamics of the usher assembly platform during uropathogenic Escherichia coli P pilus biogenesis

Uropathogenic Escherichia coli assemble surface structures termed pili or fimbriae to initiate infection of the urinary tract. P pili facilitate bacterial colonization of the kidney and pyelonephritis. P pili are assembled through the conserved chaperone-usher pathway. Much of the structural and functional understanding of the chaperone-usher pathway has been gained through investigations of type 1 pili, which promote binding to the bladder and cystitis. In contrast, the structural basis for P pilus biogenesis at the usher has remained elusive. This is in part due to the flexible and variable-length P pilus tip fiber, creating structural heterogeneity, and difficulties isolating stable P pilus assembly intermediates. Here, we circumvent these hindrances and determine cryo-electron microscopy structures of the activated PapC usher in the process of secreting two- and three-subunit P pilus assembly intermediates, revealing processive steps in P pilus biogenesis and capturing new conformational dynamics of the usher assembly machine.

Challenges in Establishing a Coronavirus Disease 2019 (COVID-19) Convalescent Plasma Donation Program in a Multicultural Environment

Context.– In the face of the coronavirus disease 2019 (COVID-19) pandemic response, it was worthwhile to test the safety and efficacy of COVID-19 convalescent plasma (CCP) transfusion. Objective.– To establish a CCP donation program based on the availability of recovered COVID-19 patients and the practical limitations to recruit clinically valid donors in a multicultural setting. Design.– From March to June 2020, we developed a program for collection of COVID-19 CCP as part of the treatment options for patients affected with COVID-19. From an initial population of 3,746 candidates, only those with positive polymerase chain reaction (PCR) results in at least two separate tests were considered. This filter reduced the eligible donor pool to 488 patients. After other exclusions were applied, such as language barrier, age, accessibility to donation, comorbidities, etc., the final count was 267 potentially eligible donors, which represented only 54.7 % (267/488) of preselected candidates. Results.– Eighty donors were called. Approximately a third of the calls provided additional challenges as outlined by the following four reasons: limited functional understanding of English; schedule availability due to rotating work timetables; transportation restrictions since public transport services were severely restricted during lockdown; and lost to follow up. Finally, a total of 38 valid donors participated, from which 45 apheresis procedures were performed. Conclusions.– As a summary of our experience, we can conclude that despite the limitations we were able to establish an effective program. A total of 90 units of CCP were collected before the pandemic curve began to flatten toward the end of June 2020.

Knowledge of preservice elementary teachers on fractions

In this paper, we studied the knowledge on didactic contents reflected by future elementary teachers when answering questions related to teaching and learning fractions. Following a qualitative methodology, specifically a case study, 9 senior pre-service elementary teachers were interviewed using a structured approach. The topic was presented to the subjects using a narrative they had previously written on how to initiate the concept of fractions with school children. After reading it, they were asked questions regarding task design, learning objectives, and mistakes and difficulties. Results identified two trends in the participants’ knowledge: a procedural or technical trend in which the stated knowledge emphasizes procedures, processes, or action modes, and a conceptual or cognitive trend in which the stated knowledge emphasizes the functional understanding of fractions and their relationships. As a conclusion, it is essential that initial teacher training emphasizes mathematical as well as didactic contents.

Structures of the ApoL1 and ApoL2 N-terminal domains reveal a non-classical four-helix bundle motif

Apolipoprotein L1 (ApoL1) is a circulating innate immunity protein protecting against trypanosome infection. However, two ApoL1 coding variants are associated with a highly increased risk of chronic kidney disease. Here we present X-ray and NMR structures of the N-terminal domain (NTD) of ApoL1 and of its closest relative ApoL2. In both proteins, four of the five NTD helices form a four-helix core structure which is different from the classical four-helix bundle and from the pore-forming domain of colicin A. The reactivity with a conformation-specific antibody and structural models predict that this four-helix motif is also present in the NTDs of ApoL3 and ApoL4, suggesting related functions within the small ApoL family. The long helix 5 of ApoL1 is conformationally flexible and contains the BH3-like region. This BH3-like α-helix resembles true BH3 domains only in sequence and structure but not in function, since it does not bind to the pro-survival members of the Bcl-2 family, suggesting a Bcl-2-independent role in cytotoxicity. These findings should expedite a more comprehensive structural and functional understanding of the ApoL immune protein family.