High above the rest: standing behaviors in the amoebae of Sappinia and Thecamoeba.

Many terrestrial microbes have evolved cell behaviors that help them rise above their substrate, often to facilitate dispersal. One example of these behaviors is found in the amoebae of Sappinia pedata, which actively lift most of their cell mass above the substrate, known as standing. This standing behavior was first described in S. pedata in the 1890s from horse dung isolates but never molecularly characterized from dung. Our study expands this understanding, revealing the first molecularly confirmed S. pedata from herbivore dung in Mississippi, USA, and describing a new species, Sappinia dangeardi n. sp., with larger trophozoite cells. Additionally, we isolated another standing amoeba, Thecamoeba homeri n. sp., from soil, exhibiting a previously unreported "doughnut shape" transient behavior. In S. dangeardi n. sp., we discovered that standing is likely triggered by substrate drying, and that actin filaments actively localize in the "stalk" to support the standing cells, as observed through confocal microscopy. While the purpose of standing behaviors has not been investigated, we hypothesize it is energetically expensive and therefore a significant evolutionary strategy in these organisms. Overall, this study emphasizes behavioral adaptations to terrestrial environments within Amoebozoa, stressing the importance of diverse laboratory conditions that replicate natural habitats.

Molecular phylogenetic analyses support the validity of Ceratiomyxa porioides (Amoebozoa, Eumycetozoa) at species level.

The frequently encountered macroscopic slime molds of the genus Ceratiomyxa have long been recognized by mycologists and protistologists for hundreds of years. These organisms are amoebozoan amoebae that live and grow inside and on the surface of decaying wood. When conditions are favorable, they form subaerial sporulating structures called fruiting bodies which take on a variety of forms. These forms are typically some arrangement of column and/or branches, but one is uniquely poroid, forming folds instead. Originally, this poroid morphology was designated as its own species. However, it was not always clear what significance fruiting body morphology held in determining species. Currently, Ceratiomyxa fruticulosa var. porioides, the poroid form, is considered a taxonomic variety of Ceratiomyxa fruticulosa based on morphological designation alone. Despite its long history of observation and study, the genus Ceratiomyxa has been paid little molecular attention to alleviate these morphological issues. We have obtained the first transcriptomes of the taxon C. fruticulosa var. porioides and found single gene phylogenetic and multigene phylogenomic support to separate it from C. fruticulosa. This provides molecular evidence that fruiting body morphology does correspond to species level diversity. Therefore, we formally raise Ceratiomyxa porioides to species level.

Bacterial cholesterol-dependent cytolysins and their interaction with the human immune response.

PURPOSE OF REVIEW: Many cholesterol-dependent cytolysin (CDC)-producing pathogens pose a significant threat to human health. Herein, we review the pore-dependent and -independent properties CDCs possess to assist pathogens in evading the host immune response. RECENT FINDINGS: Within the last 5 years, exciting new research suggests CDCs can act to inhibit important immune functions, disrupt critical cell signaling pathways, and have tissue-specific effects. Additionally, recent studies have identified a key region of CDCs that generates robust immunity, providing resources for the development of CDC-based vaccines. SUMMARY: This review provides new information on how CDCs alter host immune responses to aid bacteria in pathogenesis. These studies can assist in the design of more efficient vaccines and therapeutics against CDCs that will enhance the immune response to CDC-producing pathogens while mitigating the dampening effects CDCs have on the host immune response.

Development of the Circle of Willis Score (COWS) to help guide decision making during acute tandem occlusion treatment: Preliminary analysis.

BACKGROUND: Acute tandem occlusions (TOs) are challenging to treat. Although acute carotid stenting of the proximal lesion is well tolerated, there are certain situations when the practitioner may be wary of acute stenting (bleeding concerns). OBJECTIVE: The purpose of this study was to retrospectively study patients with tandem occlusions who had re-occlusion of the extracranial ICA and develop a Circle of Willis Score (COWS) to help predict which patients could forego acute stenting. METHODS: This is a retrospective review of TO patients with a persistent proximal occlusion following intervention (either expected or unexpected). Pre intervention CTA and intraoperative DSA were reviewed, and each patient was assigned a score 2 (complete COW), 1a (patent A1-Acomm-A1), 1p (patent Pcomm), or 0 (incomplete COW). Findings from the DSA took precedence over the CTA. Two cohorts were created, the complete COW cohort (COWS 2) versus the incomplete COW cohort (COWS 1a,1p, or 0). Angiographic outcomes were assessed using the mTICI score (2b-3) and clinical outcomes were assessed using discharge mRS (good outcome mRS 0-3). RESULTS: Of 68 TO cases, 12 had persistent proximal occlusions. There were 5/12 (42 %) patients in the complete COW cohort, and 7/12 (58 %) in the incomplete COW cohort (5/12 with scores of 1a/1p and 2/12 with a score of 0). In the complete COW cohort, there were 2 ICA-ICA and 3 ICA-MCA occlusions. In the incomplete COW cohort, there was one ICA-ICA occlusion and 6 ICA-MCA occlusions. LKW-puncture was shorter in the complete COW cohort (208 min vs. 464 min, p = 0.16). Successful reperfusion was higher in the complete COW cohort (100 % vs. 71 %). There was a trend toward better clinical outcomes in the complete COW cohort (80 % vs 29 %, p = 0.079). CONCLUSION: The COWS is a simple score that may help predict a successful clinical outcome without proximal revascularization when concerned about performing an acute carotid stent during TO treatment. Evaluation in larger TO cohort is warranted.

SAGA Complex Subunit Hfi1 Is Important in the Stress Response and Pathogenesis of Cryptococcus neoformans.

The Spt-Ada-Gcn Acetyltransferase (SAGA) complex is a highly conserved co-activator found across eukaryotes. It is composed of a number of modules which can vary between species, but all contain the core module. Hfi1 (known as TADA1 in Homo sapiens) is one of the proteins that forms the core module, and has been shown to play an important role in maintaining complex structural integrity in both brewer's yeast and humans. In this study we successfully identified the gene encoding this protein in the important fungal pathogen, Cryptococcus neoformans, and named it HFI1. The hfi1Δ mutant is highly pleiotropic in vitro, influencing phenotypes, ranging from temperature sensitivity and melanin production to caffeine resistance and titan cell morphogenesis. In the absence of Hfi1, the transcription of several other SAGA genes is impacted, as is the acetylation and deubiquination of several histone residues. Importantly, loss of the gene significantly impacts virulence in a murine inhalation model of cryptococcosis. In summary, we have established that Hfi1 modulates multiple pathways that directly affect virulence and survival in C. neoformans, and provided deeper insight into the importance of the non-enzymatic components of the SAGA complex.

Improving optical sectioning with spinning disk structured illumination microscopy.

A new fluorescence microscopy technique for optical sectioning was investigated. This technique combined Spinning Disk microscopy (SD) with Structured Illumination Microscopy (SIM), resulting in more background removal than either method. Spinning Disk Structured Illumination Microscopy (SD-SIM) resulted in higher signal-to-background ratios. The method detected and quantified a dendritic spine neck that was impossible to detect with either SIM or SD alone.

Super-Resolution Imaging of Neuronal Structures with Structured Illumination Microscopy.

Super-resolution structured illumination microscopy (SR-SIM) is an optical fluorescence microscopy method which is suitable for imaging a wide variety of cells and tissues in biological and biomedical research. Typically, SIM methods use high spatial frequency illumination patterns generated by laser interference. This approach provides high resolution but is limited to thin samples such as cultured cells. Using a different strategy for processing raw data and coarser illumination patterns, we imaged through a 150-micrometer-thick coronal section of a mouse brain expressing GFP in a subset of neurons. The resolution reached 144 nm, an improvement of 1.7-fold beyond conventional widefield imaging.

Warming may offset impact of precipitation changes on riverine nitrogen loading.

Climate change, especially in the form of precipitation and temperature changes, can alter the transformation and delivery of nitrogen on the land surface and to aquatic systems, impacting the trophic states of downstream water bodies. While the expected impacts of changes in precipitation have been explored, a quantitative understanding of the impact of temperature on nitrogen loading is lacking at landscape scales. Here, using several decades of nitrogen loading observations, we quantify how individual and combined future changes in precipitation and temperature will affect riverine nitrogen loading. We find that, contrary to recent decades, rising temperatures are likely to offset or even reverse previously reported impacts of future increases in total and extreme precipitation on nitrogen runoff across the majority of the contiguous United States. These findings highlight the multifaceted impacts of climate change on the global nitrogen cycle.

Super-resolution imaging of neuronal structure with structured illumination microscopy.

Super-resolution structured illumination microscopy (SR-SIM) is a method in optical fluorescence microscopy which is suitable for imaging a wide variety of cells and tissues in biological and biomedical research. Typically, SIM methods use high spatial frequency illumination patterns generated by laser interference. This approach provides high resolution but is limited to thin samples such as cultured cells. Using a different strategy for processing the raw data and coarser illumination patterns, we imaged through a 150 µm thick coronal section of a mouse brain expressing GFP in a subset of neurons. The resolution reached 144 nm, an improvement of 1.7 fold beyond conventional widefield imaging.

The combined effect of commercial tilapia collagen peptides and antioxidants against UV-induced skin photoaging in mice.

Chronic over-exposure to UV radiation leads to the damage of skin tissue. The aim of this study was to investigate the effects of collagen peptide (CP) and antioxidant (astaxanthin, vitamin C (Vc), and vitamin E (Ve)) combinations on skin photoaging. Forty male UV-induced BALB/c mice were randomized and fed saline or CP and antioxidants for 7 weeks using gavage feeding. The results showed that oral administration of CP, CP combined with Vc and Ve (VCE) or Haematococcus pluvialis extract (HPE) significantly (P < 0.05) reduced a* of mouse skin and increased the content of Hyp and type I collagen to varying degrees, thereby improving skin integrity. Furthermore, the combination of CP, HPE, and VCE showed increased upregulation of antioxidant enzyme expression, reduced serum ROS, and decreased inhibition of metalloproteinase expression compared to the other treatment groups. Thus, this combination showed better effects in inhibiting collagen degradation and maintaining the redox balance. The Nrf2/ARE and TGF-ß/Smad transcription systems are likely involved in these effects. Therefore, the results suggest that a diet containing CP, astaxanthin, and vitamins might be recommended to improve skin health and appearance.

Dose-dependent effects of xylooligosaccharides on glycemic regulation with L. rhamnosus CCFM1060 in diabetic mice.

Dietary intervention with the probiotic Lactobacillus rhamnosus CCFM1060 has been proved to be effective on glycemic regulation in diabetic mice. Therefore characterization of the potential symbiotic effect of prebiotic xylooligosaccharides (XOS) with L. rhamnosus CCFM1060 would be desirable. In this study, we evaluated any dose-dependent relationship between XOS and L. rhamnosus CCFM1060, and the potential impact on glycemic regulation. Diabetic mice were randomly assigned to receive 5 × 109 CFU mL-1L. rhamnosus CCFM1060, 5 × 109 CFU mL-1L. rhamnosus CCFM1060 with 250 mg kg-1 XOS (L-LXOS), or 5 × 109 CFU mL-1L. rhamnosus CCFM1060 with 500 mg kg-1 XOS (L-HXOS) for 7 weeks. In addition to characterization of the host metabolism, the intestinal microbiota were analyzed using 16S rRNA gene sequencing. The results showed that L. rhamnosus alone and L-LXOS intervention significantly alleviated diabetes symptoms and increased the populations of short-chain fatty acid (SCFA)-producing bacteria. The intake of L-HXOS had an adverse effect on glucose metabolism, causing increased insulin resistance and inflammation. Although a significant increase in the relative abundance of Bifidobacterium was observed in the L-HXOS group, the abundance of SCFA-producing bacteria, such as Romboutsia and Clostrudium sensu stricto 1, decreased. KEGG pathway analysis revealed that the adverse effects of L-HXOS intervention might be attributed to the metabolic pathways involved in amino acid, cofactor, and vitamin metabolism. This study revealed that L. rhamnosus CCFM1060 combined with different doses of XOS exerted dose-dependent effects on glucose metabolism. Therefore, the type and dose of prebiotics should be carefully evaluated when developing individualized symbiotic formula.

Effects of particle size reduction combined with ß-cyclodextrin on the in vitro dissolution and in vivo relative bioavailability of ginsenosides in Panax ginseng.

The biological effects of ginsenosides are limited by their low oral bioavailability. This study aimed to investigate the effects of particle size reduction and dispersants on the dissolution and bioavailability of ginsenosides in ginseng. Fine ginseng powder (FGP), ultrafine ginseng powder (UGP), and ultrafine ginseng powder with ß-cyclodextrin as the dispersant (UGPD) were prepared using a planetary ball mill from coarse ginseng powder (CGP, as the control). The particle size, morphology, hydration, thermal properties, and in vitro dissolution behavior of ginseng powders were characterized. The relative oral bioavailability of ginsenosides (9 protopanaxadiol (PPD)-type and 7 protopanaxatriol (PPT)-type) was determined in a rat model. Both UGP and UGPD displayed improved physiochemical properties (e.g. reduced particle size, increased hydration and thermal properties). The total in vitro dissolution of ginsenosides from UGPD was ∼17.2% higher than that from CGP; in contrast, UGP did not differ from CGP. More importantly, the in vivo pharmacokinetic study showed that the relative bioavailability of a total of 16 ginsenosides in UGPD was 180.1 ± 9.9% (CGP set as 100%), which was significantly greater than that in FGP and UGP. This suggested that dispersants were essential for preserving the benefits of ultrafine milling by preventing ultra-pulverization induced agglomeration. In particular, PPD-type ginsenosides showed similar deglycosylation trends in in vitro and in vivo experiments; in contrast, the deglycosylation states of PPT-type ginsenosides varied, which might be attributed to the relatively low abundance, glycosylation linkage, and potential involvement of the gut microbiota metabolism. Conclusively, ultrafine milling combined with a dispersant provides a simple and scalable manufacturing process that can effectively improve the bioavailability of ginseng products.

Flexible Multiplane Structured Illumination Microscope with a Four-Camera Detector.

Fluorescence microscopy provides an unparalleled tool for imaging biological samples. However, producing high-quality volumetric images quickly and without excessive complexity remains a challenge. Here, we demonstrate a four-camera structured illumination microscope (SIM) capable of simultaneously imaging multiple focal planes, allowing for the capture of 3D fluorescent images without any axial movement of the sample. This setup allows for the acquisition of many different 3D imaging modes, including 3D time lapses, high-axial-resolution 3D images, and large 3D mosaics. We imaged mitochondrial motions in live cells, neuronal structure in Drosophila larvae, and imaged up to 130 µm deep into mouse brain tissue. After SIM processing, the resolution measured using one of the four cameras improved from 357 nm to 253 nm when using a 30×/1.05 NA objective.

On the trail of iron uptake in ancestral Cyanobacteria on early Earth.

Cyanobacteria oxygenated Earth's atmosphere ~2.4 billion years ago, during the Great Oxygenation Event (GOE), through oxygenic photosynthesis. Their high iron requirement was presumably met by high levels of Fe(II) in the anoxic Archean environment. We found that many deeply branching Cyanobacteria, including two Gloeobacter and four Pseudanabaena spp., cannot synthesize the Fe(II) specific transporter, FeoB. Phylogenetic and relaxed molecular clock analyses find evidence that FeoB and the Fe(III) transporters, cFTR1 and FutB, were present in Proterozoic, but not earlier Archaean lineages of Cyanobacteria. Furthermore Pseudanabaena sp. PCC7367, an early diverging marine, benthic strain grown under simulated Archean conditions, constitutively expressed cftr1, even after the addition of Fe(II). Our genetic profiling suggests that, prior to the GOE, ancestral Cyanobacteria may have utilized alternative metal iron transporters such as ZIP, NRAMP, or FicI, and possibly also scavenged exogenous siderophore bound Fe(III), as they only acquired the necessary Fe(II) and Fe(III) transporters during the Proterozoic. Given that Cyanobacteria arose 3.3-3.6 billion years ago, it is possible that limitations in iron uptake may have contributed to the delay in their expansion during the Archean, and hence the oxygenation of the early Earth.

Abnormal larval neuromuscular junction morphology and physiology in Drosophila prickle isoform mutants with known axonal transport defects and adult seizure behavior.

Previous studies have demonstrated the striking mutational effects of the Drosophila planar cell polarity gene prickle (pk) on larval motor axon microtubule-mediated vesicular transport and on adult epileptic behavior associated with neuronal circuit hyperexcitability. Mutant alleles of the prickle-prickle (pkpk) and prickle-spiny-legs (pksple) isoforms (hereafter referred to as pk and sple alleles, respectively) exhibit differential phenotypes. While both pk and sple affect larval motor axon transport, only sple confers motor circuit and behavior hyperexcitability. However, mutations in the two isoforms apparently counteract to ameliorate adult motor circuit and behavioral hyperexcitability in heteroallelic pkpk/pksple flies. We have further investigated the consequences of altered axonal transport in the development and function of the larval neuromuscular junction (NMJ). We uncovered robust dominant phenotypes in both pk and sple alleles, including synaptic terminal overgrowth (as revealed by anti-HRP and -Dlg immunostaining) and poor vesicle release synchronicity (as indicated by synaptic bouton focal recording). However, we observed recessive alteration of synaptic transmission only in pk/pk larvae, i.e. increased excitatory junctional potential (EJP) amplitude in pk/pk but not in pk/+ or sple/sple. Interestingly, for motor terminal excitability sustained by presynaptic Ca2+ channels, both pk and sple exerted strong effects to produce prolonged depolarization. Notably, only sple acted dominantly whereas pk/+ appeared normal, but was able to suppress the sple phenotypes, i.e. pk/sple appeared normal. Our observations contrast the differential roles of the pk and sple isoforms and highlight their distinct, variable phenotypic expression in the various structural and functional aspects of the larval NMJ.

Intramolecular, Interrupted Homo-Nazarov Cascade Biscyclizations to Angular (Hetero)Aryl-Fused Polycycles.

Herein, a SnCl4 -catalyzed intramolecular, interrupted homo-Nazarov cascade biscyclization to access angular (hetero)aryl-fused polycycles is reported. Subsequent decarboxylation of the readily enolizable products afforded the angular products in up to 71 % yield over two steps, with the trans-diastereomers as the major products. The cyclopropyl homo-Nazarov cyclization precursors were formed using a scalable and modular synthetic route that, ultimately, offers access to 6,6,6-, 6,6,5-, 6,5,6-, 6,6,5,6-, and 6,6,6,5-fused angular polycyclic products. To showcase the rigor and utility of the method, an 8-step total synthesis of (±)-1-oxoferruginol, an antibacterial aromatic abietane diterpenoid, was disclosed.

Optimization of laser deposited silver nanoparticle substrates for surface-enhanced raman spectroscopy.

Creating sensitive and reproducible substrates for surface-enhanced Raman spectroscopy (SERS) has been a challenge in recent years. While SERS offers significant benefits over traditional Raman spectroscopy, certain hindrances have limited their commercial use, especially in settings where low limits of detection are necessary. We studied a variety of laser-deposited silver microstructured SERS substrates with different morphology as a means to optimize analyte detection. We found that using a 405 nm laser to deposit lines of silver nanoparticles (AgNPS) from a 2 mM silver nitrate and sodium citrate solution offered not only the best enhancement, but also the most consistent and reproducible substrates. We also found that the probability of deposition by laser was wavelength dependent and that longer wavelengths were less likely to deposit than shorter wavelengths. This work offers a better understanding of the laser deposition process as well as how substrate shape and structure effect SERS signals.

Compartment-specific tuning of dendritic feature selectivity by intracellular Ca2+ release.

Dendritic calcium signaling is central to neural plasticity mechanisms that allow animals to adapt to the environment. Intracellular calcium release (ICR) from the endoplasmic reticulum has long been thought to shape these mechanisms. However, ICR has not been investigated in mammalian neurons in vivo. We combined electroporation of single CA1 pyramidal neurons, simultaneous imaging of dendritic and somatic activity during spatial navigation, optogenetic place field induction, and acute genetic augmentation of ICR cytosolic impact to reveal that ICR supports the establishment of dendritic feature selectivity and shapes integrative properties determining output-level receptive fields. This role for ICR was more prominent in apical than in basal dendrites. Thus, ICR cooperates with circuit-level architecture in vivo to promote the emergence of behaviorally relevant plasticity in a compartment-specific manner.