Clustering of rRNA operons in E. coli is disrupted by σH.

Chromosomal organization in E. coli as examined by Hi-C methodology indicates that long-range interactions are sparse. Yet, spatial co-localization or 'clustering' of 6/7 ribosomal RNA (rrn) operons distributed over half the 4.6 Mbp genome has been captured by two other methodologies - fluorescence microscopy and Mu transposition. Our current understanding of the mechanism of clustering is limited to mapping essential cis elements. To identify trans elements, we resorted to perturbing the system by chemical and physical means and observed that heat shock disrupts clustering. Levels of σH are known to rise as a cellular response to the shock. We show that elevated expression of σH alone is sufficient to disrupt clustering, independent of heat stress. The anti-clustering activity of σH does not depend on its transcriptional activity but requires core-RNAP interaction and DNA-binding activities. This activity of σH is suppressed by ectopic expression of σD suggesting a competition for core-RNAP. A query of the other five known σ factors of E. coli found that elevated expression of FecI, the ECF σ factor that controls iron citrate transport, also perturbs clustering and is also suppressed by σD. We discuss a possible scenario for how these membrane-associated σ factors participate in clustering of distant rrn loci.

High-Throughput Mapping of Chromosomal Conformations in E. coli Under Physiological Conditions Using Massively Multiplexed Mu Transposition.

Many approaches for measuring three-dimensional chromosomal conformations rely upon formaldehyde crosslinking followed by subsequent proximity ligation, a family of methods exemplified by 3C, Hi-C, etc. Here we provide an alternative crosslinking-free procedure for high-throughput identification of long-range contacts in the chromosomes of enterobacteria, making use of contact-dependent transposition of phage Mu to identify distant loci in close contact. The procedure described here will suffice to provide a comprehensive map of transposition frequencies between tens of thousands of loci in a bacterial genome, with the resolution limited by the diversity of the insertion site library used and the sequencing depth applied.

Efflux-linked accelerated evolution of antibiotic resistance at a population edge.

Efflux is a common mechanism of resistance to antibiotics. We show that efflux itself promotes accumulation of antibiotic-resistance mutations (ARMs). This phenomenon was initially discovered in a bacterial swarm where the linked phenotypes of high efflux and high mutation frequencies spatially segregated to the edge, driven there by motility. We have uncovered and validated a global regulatory network connecting high efflux to downregulation of specific DNA-repair pathways even in non-swarming states. The efflux-DNA repair link was corroborated in a clinical "resistome" database: genomes with mutations that increase efflux exhibit a significant increase in ARMs. Accordingly, efflux inhibitors decreased evolvability to antibiotic resistance. Swarms also revealed how bacterial populations serve as a reservoir of ARMs even in the absence of antibiotic selection pressure. High efflux at the edge births mutants that, despite compromised fitness, survive there because of reduced competition. This finding is relevant to biofilms where efflux activity is high.

Goodbye PAM: Phage λ's Red recombination system cripples PAMs and helps dodge CRISPR attacks.

Ever wondered how the phage λ Red recombination system resembles the Red Queen? Hossain et al. (2021) provide an answer in this issue of Cell Host & Microbe. They show that Red debilitates PAM sequences by mutagenic repair of CRISPR-targeted DNA breaks in infecting λ, thus shaping the phage-CRISPR arms race.

Loss-of-Function Mutations in epaR Confer Resistance to ϕNPV1 Infection in Enterococcus faecalis OG1RF.

Enterococcus faecalis is a Gram-positive opportunistic pathogen that inhabits the human gastrointestinal tract. Because of the high frequency of antibiotic resistance among Enterococcus clinical isolates, interest in using phage to treat enterococcal infections and to decolonize high-risk patients for antibiotic-resistant Enterococcus is rising. Bacteria can evolve phage resistance, but there is little published information on these mechanisms in E. faecalis In this report, we identified genetic determinants of E. faecalis resistance to phage NPV1 (ϕNPV1). We found that loss-of-function mutations in epaR confer ϕNPV1 resistance by blocking phage adsorption. We attribute the inability of the phage to adsorb to the modification or loss of an extracellular polymer in strains with inactivated epaR Phage-resistant epaR mutants exhibited increased daptomycin and osmotic stress susceptibilities. Our results demonstrate that in vitro spontaneous resistance to ϕNPV1 comes at a cost in E. faecalis OG1RF.

Activated neural stem cells contribute to stroke-induced neurogenesis and neuroblast migration toward the infarct boundary in adult rats.

Stroke increases neurogenesis. The authors investigated whether neural stem cells or progenitor cells in the adult subventricular zone (SVZ) of rats contribute to stroke-induced increase in neurogenesis. After induction of stroke in rats, the numbers of cells immunoreactive to doublecortin, a marker for immature neurons, increased in the ipsilateral SVZ and striatum. Infusion of an antimitotic agent (cytosine-beta-D-arabiofuranoside, Ara-C) onto the ipsilateral cortex eliminated more than 98% of actively proliferating cells in the SVZ and doublecortin-positive cells in the ipsilateral striatum. However, doublecortin-positive cells rapidly replenished after antimitotic agent depletion of actively proliferating cells. Depleting the numbers of actively proliferating cells in vivo had no effect on the numbers of neurospheres formed in vitro, yet the numbers of neurospheres derived from stroke rats significantly (P<0.05) increased. Neurospheres derived from stroke rats self-renewed and differentiated into neurons and glia. In addition, doublecortin-positive cells generated in the SVZ migrated in a chainlike structure toward ischemic striatum. These findings indicate that in the adult stroke brain, increases in recruitment of neural stem cells contribute to stroke-induced neurogenesis, and that newly generated neurons migrate from the SVZ to the ischemic striatum.

Intraspinal endodermal cyst: ultrastructural study of abnormal cilia.

Abnormal cilia were demonstrated in the lining epithelial cells of three cases of intraspinal endodermal (bronchogenic) cyst. The changes comprised a wide spectrum of ultrastructural abnormalities, including (a). cilia with abnormal axonemal microtubules, (b). swollen cilia, (c). compound cilia with or without excessive ciliary matrix, (d). naked cilia without limiting membrane, and (e). intracytoplasmic cilia and aggregates of microtubules. Of these, compound cilia and swollen cilia were most common. Cilia with dynein arm deficiency were not observed. Ciliary abnormalities found in the present study were very similar to those described in the bronchial epithelium of various diseases. The present findings suggest that the lining epithelium of intraspinal endodermal cyst shares similar ciliogenesis and susceptibility to abnormal ciliary formation as that of the bronchial epithelium.

Magnetic resonance imaging and neurosphere therapy of stroke in rat.

We intracisternally transplanted subventricular zone (SVZ) cells labeled by ferromagnetic particles into stroked rats. Migration of transplanted cells was non-invasively tracked using magnetic resonance imaging (MRI). We found that transplanted cells selectively migrated towards the ischemic parenchyma at a mean speed of 65 +/- 14.6 microm/hr in living rats. Migration of transplanted cells in the brain was also measured histopathologically. Rats transplanted with SVZ cells exhibited significant improvement of neurological function. Our data suggest that intracisternal transplantation of SVZ cells provides an avenue for cell therapy of stroke and that MRI can be used to track grafted cells in the brain.

Post-radiation dedifferentiation of meningioma into osteosarcoma.

BACKGROUND: A number of osteoblastic meningiomas, primary osteosarcomas of the meninges, and post-radiation osteosarcomas of the head have been reported. However, postradiation dedifferentiation of meningioma into osteosarcoma has not been reported previously. CASE PRESENTATION: In 1987 a caucasian man, then 38 years old, presented with a pituitary macroadenoma. He underwent a subtotal resection of the tumor and did well until 1990 when a recurrent tumor was diagnosed. This was treated with subtotal resection of the tumor, followed by radiation therapy for six weeks to a total of 54 Gy. He was considered "disease-free" for nearly ten years. However, most recently in July 2000, he presented with a visual field deficit due to a second recurrence of his pituitary macroadenoma, now with suprasellar extension. At this time, as an incidental finding, a mass attached to the dura was noted in the left parietal hemisphere. This dura-based mass had grown rapidly by January 2001 and was excised. It showed histological, immunohistochemical, and electron microscopic features of malignant meningioma and osteosarcoma with a sharp demarcation between the two components. CONCLUSIONS: We report a rare case of a radiation induced dedifferentiation of meningioma into osteosarcoma, which has not been reported previously.

Lateral supratentorial endodermal cyst: case report and review of literature.

OBJECTIVE AND IMPORTANCE: Epithelial cysts of the central nervous system are unusual entities, with the majority reported to occur in the spinal canal. More unusual is the intracranial presentation, which shows a predilection for midline localization in the posterior fossa, brainstem, and suprasellar regions. This report discusses the differential diagnosis, pathogenesis, radiographic presentation, and therapeutic considerations of a laterally positioned cerebral convexity endodermal cyst. CLINICAL PRESENTATION: A 49-year-old right-handed man presented with approximately a 30-month history of short- and long-term memory difficulties. A magnetic resonance imaging study revealed a large, nonenhancing, extra-axial cystic lesion overlying the right lateral frontal convexity. INTERVENTION: A right frontoparietal craniotomy exposed a large extra-axial cyst with an opaque, yellowish-white membrane containing a mucoid fluid. Histological analysis disclosed a layer of unremarkable, ciliated columnar epithelium with a basement membrane that stained positive for cytokeratin, periodic acid-Schiff, and Alcian blue. No evidence of either a muscular or cartilaginous layer around the mucosa was present to further delineate neurenteric versus bronchogenic origin of the cyst. CONCLUSION: This case involved the occurrence of a solitary endodermal cyst as an extra-axial mass localized over the lateral frontal lobe. The lateral supratentorial localization of this lesion illustrates the need for consideration of the pathogenesis of this entity as well as its diagnostic differentiation from other cystic abnormalities in this region. A review of the histochemistry of endodermal, neuroepithelial, and other cerebral cysts is presented.

Focal cerebral ischemia in rats produced by intracarotid embolization with viscous silicone.

Many factors contribute to the severity of neuronal cell death and the functional outcome in stroke. We describe an embolic model of focal cerebral ischemia in the rat that does not require craniotomy and is compatible with continuous measurement of regional CBF using multichannel laser Doppler flow (LDF) technique. Either a 22 microliters (large lesion) or 11 microliters (small lesion) bolus of viscous silicone was injected cephalad into the internal carotid artery. Upon injection, LDF decreased abruptly, most severely in the parietal cortex (-74% +/- 5%) in the large lesion and in the occipital cortex (-69% +/- 10%) in the small lesion model. Over the first hour, post-embolization LDF improved in most areas (e.g. -48% +/- 9% parietal, large lesion) but declined in the small lesion group in the occipital region (-81% +/- 8%). CBF measured by [C]14-IAP autoradiography 1 h post-embolization in the large lesion model demonstrated near-hemispheric ischemia (70% of hemisphere) with sparing of cingulate cortex. Autoradiography demonstrated that ischemia in the small lesion was largely cortical. Light microscopy of brains embolized with 11 microliters of dyed silicone showed filling of pial vessels with no silicone in the Circle of Willis or parenchyma. No animals in the large lesion group survived 24 h. Thirteen of 15 animals in the small lesion group survived for two weeks with resolution of initial hemiplegia, ocular asymmetry and weight loss. Hematoxylin-eosin staining two weeks post-embolization showed signs of severe hypoxia and infarction. In conclusion, the intracarotid silicone embolization technique produces a titrable, reproducible permanent ischemic injury by blocking perfusion in the pial circulation, and is amenable to multisite monitoring with laser Doppler flowmetry. The smaller embolus produces cortical infarction with high rate of survival and neurological recovery.