CetZ tubulin-like proteins control archaeal cell shape.

Tubulin is a major component of the eukaryotic cytoskeleton, controlling cell shape, structure and dynamics, whereas its bacterial homologue FtsZ establishes the cytokinetic ring that constricts during cell division. How such different roles of tubulin and FtsZ evolved is unknown. Studying Archaea may provide clues as these organisms share characteristics with Eukarya and Bacteria. Here we report the structure and function of proteins from a distinct family related to tubulin and FtsZ, named CetZ, which co-exists with FtsZ in many archaea. CetZ X-ray crystal structures showed the FtsZ/tubulin superfamily fold, and one crystal form contained sheets of protofilaments, suggesting a structural role. However, inactivation of CetZ proteins in Haloferax volcanii did not affect cell division. Instead, CetZ1 was required for differentiation of the irregular plate-shaped cells into a rod-shaped cell type that was essential for normal swimming motility. CetZ1 formed dynamic cytoskeletal structures in vivo, relating to its capacity to remodel the cell envelope and direct rod formation. CetZ2 was also implicated in H. volcanii cell shape control. Our findings expand the known roles of the FtsZ/tubulin superfamily to include archaeal cell shape dynamics, suggesting that a cytoskeletal role might predate eukaryotic cell evolution, and they support the premise that a major function of the microbial rod shape is to facilitate swimming.

Complete genome sequence of Helicobacter pylori B128 7.13 and a single-step method for the generation of unmarked mutations.

BACKGROUND: Helicobacter pylori represents an interesting model of bacterial pathogenesis given that most infections are asymptomatic, while a minority of infections cause severe gastric disease. H pylori strain B128 7.13 is used extensively to understand H pylori pathophysiology. Due to extensive restriction-modification systems, the fact that only some H pylori strains are naturally transformable, the inability of common plasmid and transposon vectors to replicate in this bacterium, as well as the limited number of antibiotic cassettes that are functional in H pylori, there are relatively few genetic tools for the mutagenesis of this bacterium. MATERIALS AND METHODS: Here, we use PacBio and Illumina sequencing to reveal the complete genome sequence of H pylori B128 7.13. Furthermore, we describe a system to generate markerless and scarless mutations on the H pylori chromosome using the counter-selection marker, galactokinase from Escherichia coli. RESULTS: We show that this mutagenesis strategy can be used to generate in-frame insertions, gene deletions, and multiple independent mutations in B128 7.13. Using the closed genome as a reference, we also report the absence of second site chromosomal mutations and/or rearrangements in our mutagenized strains. We compare the genome sequence of H pylori B128 7.13 with a closely related strain, H pylori B8, and reveal one notable region of difference, which is a 1430 bp insertion encoding a H pylori-specific DUF874 family protein of unknown function. CONCLUSIONS: This article reports the closed genome of the important H pylori B128 7.13 strain and a mutagenesis method that can be adopted by researchers as an alternative strategy to generate isogenic mutants of H pylori in order to further our understanding of this bacterium.

The improving outcomes in intermittent exotropia study: outcomes at 2 years after diagnosis in an observational cohort.

BACKGROUND: The purpose of this study was to investigate current patterns of management and outcomes of intermittent distance exotropia [X(T)] in the UK. METHODS: This was an observational cohort study which recruited 460 children aged < 12 years with previously untreated X(T). Eligible subjects were enrolled from 26 UK hospital ophthalmology clinics between May 2005 and December 2006. Over a 2-year period of follow-up, clinical data were prospectively recorded at standard intervals from enrolment. Data collected included angle, near stereoacuity, visual acuity, control of X(T) measured with the Newcastle Control Score (NCS), and treatment. The main outcome measures were change in clinical outcomes (angle, stereoacuity, visual acuity and NCS) in treated and untreated X(T), 2 years from enrolment (or, where applicable, 6 months after surgery). Change over time was tested using the chi-square test for categorical, Wilcoxon test for non-parametric and paired-samples t-test for parametric data. RESULTS: At follow-up, data were available for 371 children (81% of the original cohort). Of these: 53% (195) had no treatment; 17% (63) had treatment for reduced visual acuity only (pure refractive error and amblyopia); 13% (50) had non surgical treatment for control (spectacle lenses, occlusion, prisms, exercises) and 17% (63) had surgery. Only 0.5% (2/371) children developed constant exotropia. The surgically treated group was the only group with clinically significant improvements in angle or NCS. However, 8% (5) of those treated surgically required second procedures for overcorrection within 6 months of the initial procedure and at 6-month follow-up 21% (13) were overcorrected. CONCLUSIONS: Many children in the UK with X(T) receive active monitoring only. Deterioration to constant exotropia, with or without treatment, is rare. Surgery appears effective in improving angle of X(T) and NCS, but rates of overcorrection are high.

Spatial regulation of cell motility and its fitness effect in a surface-attached bacterial community.

On a surface, microorganisms grow into a multi-cellular community. When a community becomes densely populated, cells migrate away to expand the community's territory. How microorganisms regulate surface motility to optimize expansion remains poorly understood. Here, we characterized surface motility of Proteus mirabilis. P. mirabilis is well known for its ability to expand its colony rapidly on a surface. Cursory visual inspection of an expanding colony suggests partial migration, i.e., one fraction of a population migrates while the other is sessile. Quantitative microscopic imaging shows that this migration pattern is determined by spatially inhomogeneous regulation of cell motility. Further analyses reveal that this spatial regulation is mediated by the Rcs system, which represses the expression of the motility regulator (FlhDC) in a nutrient-dependent manner. Alleviating this repression increases the colony expansion speed but results in a rapid drop in the number of viable cells, lowering population fitness. These findings collectively demonstrate how Rcs regulates cell motility dynamically to increase the fitness of an expanding bacterial population, illustrating a fundamental trade-off underlying bacterial colonization of a surface.

Stochastic activation of a family of TetR type transcriptional regulators controls phenotypic heterogeneity in Acinetobacter baumannii.

Phenotypic heterogeneity is an important mechanism for regulating bacterial virulence, where a single regulatory switch is typically activated to generate virulent and avirulent subpopulations. The opportunistic pathogen Acinetobacter baumannii can transition at high frequency between virulent opaque (VIR-O) and avirulent translucent subpopulations, distinguished by cells that form opaque or translucent colonies. We demonstrate that expression of 11 TetR-type transcriptional regulators (TTTRs) can drive cells from the VIR-O opaque subpopulation to cells that form translucent colonies. Remarkably, in a subpopulation of VIR-O cells, four of these TTTRs were stochastically activated in different combinations to drive cells to the translucent state. The resulting translucent subvariants exhibited unique phenotypic differences and the majority were avirulent. Due to their functional redundancy, a quadruple mutant with all four of these TTTRs inactivated was required to observe a loss of switching from the VIR-O state. Further, we demonstrate a small RNA, SrvS, acts as a "rheostat," where the levels of SrvS expression influences both the VIR-O to translucent switching frequency, and which TTTR is activated when VIR-O cells switch. In summary, this work has revealed a new paradigm for phenotypic switching in bacteria, where an unprecedented number of related transcriptional regulators are activated in different combinations to control virulence and generate unique translucent subvariants with distinct phenotypic properties.

Observing Bacterial Persistence at Single-Cell Resolution.

Within a bacterial population, there can be a subpopulation of cells with an antibiotic-tolerant persister phenotype characterized by long lag phase. Their long lag phase necessitates long (hours or days) periods of single-cell observation to capture high-quality quantitative information about persistence. We describe a method of single-cell imaging using glass bottom dishes and a nutrient agarose pad that allows for long-term single-cell microscopy observation in a stable environment. We apply this method to characterize the lag phase and persistence of individual Escherichia coli cells.

Stable retention of chloramphenicol-resistant mtDNA to rescue metabolically impaired cells.

The permanent transfer of specific mtDNA sequences into mammalian cells could generate improved models of mtDNA disease and support future cell-based therapies. Previous studies documented multiple biochemical changes in recipient cells shortly after mtDNA transfer, but the long-term retention and function of transferred mtDNA remains unknown. Here, we evaluate mtDNA retention in new host cells using 'MitoPunch', a device that transfers isolated mitochondria into mouse and human cells. We show that newly introduced mtDNA is stably retained in mtDNA-deficient (ρ0) recipient cells following uridine-free selection, although exogenous mtDNA is lost from metabolically impaired, mtDNA-intact (ρ+) cells. We then introduced a second selective pressure by transferring chloramphenicol-resistant mitochondria into chloramphenicol-sensitive, metabolically impaired ρ+ mouse cybrid cells. Following double selection, recipient cells with mismatched nuclear (nDNA) and mitochondrial (mtDNA) genomes retained transferred mtDNA, which replaced the endogenous mutant mtDNA and improved cell respiration. However, recipient cells with matched mtDNA-nDNA failed to retain transferred mtDNA and sustained impaired respiration. Our results suggest that exogenous mtDNA retention in metabolically impaired ρ+ recipients depends on the degree of recipient mtDNA-nDNA co-evolution. Uncovering factors that stabilize exogenous mtDNA integration will improve our understanding of in vivo mitochondrial transfer and the interplay between mitochondrial and nuclear genomes.

Pressure-Driven Mitochondrial Transfer Pipeline Generates Mammalian Cells of Desired Genetic Combinations and Fates.

Generating mammalian cells with desired mitochondrial DNA (mtDNA) sequences is enabling for studies of mitochondria, disease modeling, and potential regenerative therapies. MitoPunch, a high-throughput mitochondrial transfer device, produces cells with specific mtDNA-nuclear DNA (nDNA) combinations by transferring isolated mitochondria from mouse or human cells into primary or immortal mtDNA-deficient (ρ0) cells. Stable isolated mitochondrial recipient (SIMR) cells isolated in restrictive media permanently retain donor mtDNA and reacquire respiration. However, SIMR fibroblasts maintain a ρ0-like cell metabolome and transcriptome despite growth in restrictive media. We reprogrammed non-immortal SIMR fibroblasts into induced pluripotent stem cells (iPSCs) with subsequent differentiation into diverse functional cell types, including mesenchymal stem cells (MSCs), adipocytes, osteoblasts, and chondrocytes. Remarkably, after reprogramming and differentiation, SIMR fibroblasts molecularly and phenotypically resemble unmanipulated control fibroblasts carried through the same protocol. Thus, our MitoPunch "pipeline" enables the production of SIMR cells with unique mtDNA-nDNA combinations for additional studies and applications in multiple cell types.

Intracellular Photothermal Delivery for Suspension Cells Using Sharp Nanoscale Tips in Microwells.

Efficient intracellular delivery of biomolecules into cells that grow in suspension is of great interest for biomedical research, such as for applications in cancer immunotherapy. Although tremendous effort has been expended, it remains challenging for existing transfer platforms to deliver materials efficiently into suspension cells. Here, we demonstrate a high-efficiency photothermal delivery approach for suspension cells using sharp nanoscale metal-coated tips positioned at the edge of microwells, which provide controllable membrane disruption for each cell in an array. Self-aligned microfabrication generates a uniform microwell array with three-dimensional nanoscale metallic sharp tip structures. Suspension cells self-position by gravity within each microwell in direct contact with eight sharp tips, where laser-induced cavitation bubbles generate transient pores in the cell membrane to facilitate intracellular delivery of extracellular cargo. A range of cargo sizes were tested on this platform using Ramos suspension B cells with an efficiency of >84% for Calcein green (0.6 kDa) and >45% for FITC-dextran (2000 kDa), with retained viability of >96% and a throughput of >100 000 cells delivered per minute. The bacterial enzyme ß-lactamase (29 kDa) was delivered into Ramos B cells and retained its biological activity, whereas a green fluorescence protein expression plasmid was delivered into Ramos B cells with a transfection efficiency of >58%, and a viability of >89% achieved.

Use of the combined recession and resection of a rectus muscle procedure in the management of incomitant strabismus.

BACKGROUND: Scott described a method of achieving the effect of a posterior fixation procedure by combining resection and recession of a rectus muscle, while maintaining the ability to adjust primary position alignment with adjustable sutures. METHODS: A retrospective review of combined recession-resection procedures on rectus muscles between 1998 and 2002 was carried out. RESULTS: We identified 22 patients, 17 females and 5 males. The mean age at presentation was 44 years. The etiology was felt to be paralytic in seven patients (31.8%), mechanical/restrictive in seven patients (31.8%), and due to residual childhood strabismus in eight patients (36.4%). Twenty patients had undergone previous strabismus surgery, ranging from one to six operations (mean, 1.75). Scott procedures were performed on 25 rectus muscles of 22 patients; 12 on the lateral rectus muscles, 7 on the inferior rectus muscles, 3 on the medial rectus muscles, and 3 on the superior rectus muscles. All but one patient had a measurable improvement in gaze incomitance, and 11 of 12 who had a measurement of the field of binocular single vision showed improvement. Twenty patients had follow-up periods of 3 months or more, with a range of 3 to 30 months and a mean of 9.35 months. CONCLUSIONS: The combined recession-resection procedure advocated by Scott has a role in the management of incomitant strabismus.

Botulinum toxin in the management of internuclear ophthalmoplegia.

BACKGROUND: Internuclear ophthalmoplegia (INO) presents as a complex ocular motility problem due to medial longitudinal fasciculus damage. The symptoms are disabling and challenging to manage. We evaluate the efficacy of botulinum toxin in the management of INO. METHODS: A retrospective review of 16 patients with INO managed by botulinum toxin A injection into one or more extraocular muscles over the past 20 years was performed. The age, sex, etiology, symptoms, angle of deviation, and binocular function pre- and postinjection were analyzed. RESULTS: The mean age at presentation was 45.1 years with a male/female ratio of 9:7. The most common etiologies included brainstem hemorrhage in five and multiple sclerosis in four. After injection diplopia reduced in 14 (87.5%), appearance improved in 9 (56.3%), and the head posture improved in 4 (25%). Convergence recovered in two patients (12.5%) and stereopsis improved in three patients (18.8%). Complications included transient ptosis in one patient, and one patient had an overcorrection. Two patients complained of transient vertical diplopia that resolved within 3 weeks. At final follow-up, five (31.5%) patients continued on maintenance injections, three (18.8%) were stable and discharged, while the rest were managed by occlusion (five), prisms (one), or surgery (two). CONCLUSIONS: We report on the management of INO by botulinum toxin. Benefit was noted in terms of reduced diplopia and occasionally improved binocular function. Though the benefits are limited by the need for repeated injections, for patients with this debilitating condition, botulinum toxin provides some symptomatic relief.

Strabismus in adults with uveal melanoma following episcleral plaque brachytherapy.

PURPOSE: To ascertain the incidence of persistent strabismus in patients treated with plaque brachytherapy and its subsequent treatment. METHODS: A single center retrospective case note review of adult patients with persistent diplopia or strabismus following plaque brachytherapy for all types of intraocular tumors between 1996 and 2004. RESULTS: A total of 929 consecutive adults underwent plaque brachytherapy during the study period at a single center. Sixteen patients (1.7%) with treated uveal melanoma developed persistent diplopia or strabismus. In 11 patients (69%) the timing of onset was in the first year, in 2 (13%) in the second year, and one each (6% each) in years 5, 7, and 8. Two patients (13%) did not require any intervention. Fourteen patients (88%) required treatment: 7 (50%) were treated with prisms only, 3 (21%) underwent botulinum toxin (BTXA) injections, and 4 (29%) were treated with extraocular muscle surgery (3 required one operation and one required 2 procedures). CONCLUSIONS: The incidence of ocular motility disorders following plaque brachytherapy in our cohort was 1.7% over 8 years and we include this in the consent process for conservative treatment of intraocular tumors. Options for treatment for persistent diplopia or strabismus include prisms, botulinum toxin injection, or surgery.

Full-tendon nasal transposition of the vertical rectus muscles: a retrospective review.

The authors report the results of a retrospective review of patients who underwent nasal transposition of the vertical rectus muscles between 1997 and 2004. Eight patients were identified, 4 males and 4 females. There was an average age at surgery of 37 years, with a range from 8 to 79 years. The aetiologies included 6 patients with trauma to their medial rectus (4 following endoscopic sinus surgery) and 2 patients with paralytic medial rectus muscles secondary to 3rd nerve palsy. All patients underwent whole tendon transposition of the superior and inferior rectus muscles, with resection of both muscles in 7 cases, before reattachment adjacent to the upper and lower borders of the medial rectus, respectively. One patient had a reduced amount of resection and this was combined with inferior oblique disinsertion and traction sutures. All patients had a reduction in deviation in the primary position and in 5 patients there was some improvement in adduction. A consequence of surgery was a degree of limitation of abduction, elevation and depression in some patients. Overall, patients were satisfied with the improvement in their appearance.

Benefits of retroequatorial four horizontal muscle recession surgery in congenital idiopathic nystagmus in adults.

INTRODUCTION: Four muscle surgery for congenital nystagmus was originally described in 1956 and popularized by two articles in the 1990s. A review of the literature revealed only three studies of seven, nine, and six patients published since then. We therefore reviewed our patients to determine the objective and subjective benefits of this procedure. METHODS: A retrospective review was carried out on patients who underwent bilateral maximal horizontal muscle recessions for congenital nystagmus between 1997 and 2002. Improvement in visual acuity was documented. An external assessor administered an anonymized questionnaire to discover any perceived benefit. Eighteen patients were identified, 12 men and 6 women, with an average age at surgery of 32 years. RESULTS: Preoperative visual acuity ranged from 6/9 binocularly to 6/60. All underwent bilateral medial rectus muscle recessions of 8 mm to 10 mm and bilateral lateral rectus muscle recessions of 8 mm to 12 mm. Nine patients (50%) gained one line of Snellen visual acuity. There were four complications: one scleral perforation; two developed exotropia; and one complained of asthenopic symptoms. This latter patient was the only one who experienced some deterioration of vision acuity. Fourteen of 18 (78%) questionnaires were returned. Eight patients said they were pleased; four were indifferent and two were displeased. CONCLUSIONS: We conclude that this operation delivers limited objective benefit. None of our patients were able to obtain a driving license. Fifty-seven percent of patients expressed the view that they were glad to have had the surgery, although the visual improvement was only slight.

Does botulinum toxin have a role in the treatment of secondary strabismus?*.

METHODS: We reviewed the Toxin Clinic database over a 20-year period, identifying patients with strabismus secondary to permanent or temporary impairment of vision. RESULTS: Five hundred-and-three patients who fulfilled our criteria were treated with Botulinum toxin. There were 301 males and 202 females, with a mean age at treatment of 35 years (range 6-86). Three hundred-and-eighty-three patients (76%) had exotropia, 114 (22.5%) had esotropia and 6 (1.5%) had vertical strabismus. Reduced vision secondary to trauma accounted for 45% (227) of patients. Other causes were congenital anomalies 121 (24%), refractive error 30 (6%), retinal detachment 20 (4%), senile cataract 24 (5%), corneal problems 19 (4%), glaucoma 10 (2%) and other 52 (10%). One hundred-and-ninety-three (38%) were aphakic. One thousand-four-hundred-and-fifty-seven injections were given, with a range of 1-50 per patient. Ninety-nine (20%) patients were managed with continued toxin treatment. Two hundred-and-eighteen patients (43%) proceeded to surgery. Forty patients (8%) required no further treatment as they regained binocularity by injection alone. CONCLUSIONS: Botulinum toxin appears to have a role in the treatment of secondary strabismus, since only 3% (14) failed to obtain any reduction in their angle. It was well tolerated, with no permanent side effects. Only 22 (4%) patients developed ptosis.

Radiotherapy in thyroid eye disease: the effect on the field of binocular single vision.

PURPOSE: Thyroid eye disease (TED) can be a functionally disabling condition if ocular muscle involvement causes diplopia. The extraocular muscle restriction creates a reduced or eccentric field of binocular single vision (BSV). Orbital radiotherapy is now widely used in the treatment of TED, and although it has been reported as improving ocular motility, there have been few quantitative studies of the effect of treatment on ocular motor function. METHODS: Retrospective case note review of patients undergoing orbital radiotherapy for TED between 1992-1998 identified 79 case records. A total of 27 patients had diplopia in primary position or a significantly reduced binocular field before undergoing radiotherapy. The fields of BSV were analyzed pretreatment and at 3, 12, and 24 months after therapy to assess any improvement in function. We used the field of BSV as an outcome measure because it can be quantified and is a good indicator of functional ability. RESULTS: None of the 12 patients with double vision in primary position pretreatment regained a central binocular field with radiotherapy alone. Of the 15 patients with a central but reduced binocular field, 8 (53%) remained unchanged with treatment. In 4 patients (26.6%), there was an improvement in the field, while in 3 (20%) the field deteriorated. In all, 12 patients (44%) went on to require strabismus surgery. CONCLUSIONS: Orbital radiotherapy alone is ineffective in treating restrictive thyroid myopathy and improving binocular function.

Adherence syndrome following inferior oblique surgery: management and outcome of 14 cases.

BACKGROUND: Adherence Syndrome is diagnosed by limited upgaze, usually in the presence of an ipsilateral consecutive hypotropia in primary position, which is worse in lateral gaze. There is also a positive forced duction test. METHODS: A retrospective review of patients diagnosed with sdherence syndrome following strabismus surgery under the care of a single ophthalmologist between 1989 and 2001 was performed. Fourteen patients were identified. All patients were thought to have undergone previous ipsilateral inferior oblique surgery, which was felt to be the cause of the restriction. RESULTS: We usually performed an extensive conjunctival peritomy over nearly 180 degrees, recessing the conjunctiva while leaving the sclera to re-epithelialise, and in addition recessed the inferior rectus by 2.5-3 mm. Only one patient required an additional operation. Of the 11 patients who had undergone surgery at our institution all showed a significant improvement in their symptoms. five were discharged happy, three continue to be followed and three failed to attend further appointments after their initial visit two weeks post-operation. CONCLUSIONS: It is not possible to restore normal eye movements in these patients. Our results show that one can improve the appearance in primary position with single vision. Patients should be given realistic expectations before surgery and the limited aims of surgery must be explained.

Modified Harada-Ito procedure in bilateral superior oblique paresis.

BACKGROUND: Patients with bilateral superior oblique palsies may complain of diplopia and torsion, especially in downgaze. They are difficult to manage surgically, although bilateral modified Harada-Ito procedures may be of value. METHODS: A retrospective review was carried out of patients treated with bilateral Fells' modified Harada-Ito procedures for bilateral superior oblique palsy between 1989 and March 2000, the patients being identified from operating records. RESULTS: Twenty-three patients were identified (9 females and 14 males) with a mean age at presentation of 37 years (range 19 to 75). In 20 cases (87%) the aetiology was trauma (14 due to traffic accidents). All patients had diplopia at presentation (3 specifically mentioned torsion) with a mean duration of 22.5 months. Eleven patients had abnormal head postures and 13 were binocular. Mean vertical deviation was 3.2(delta)for distance and 4(delta)for near. Maximum torsion ranged from 5 degrees (previous surgery) to 24 degrees excylotorsion with a mean of 13 degrees. Thirteen patients had a V pattern. After surgery the mean vertical deviation was 1.5(delta )for distance and 1(delta)for near. Ten patients were symptom free, 8 had reduced diplopia and 5 were unchanged. The worst torsion post-op ranged from 1 degrees of intorsion to 11 degrees of excyclotorsion (mean 4 degrees ). Further vertical muscle surgery was required in 10 (43%) patients. Mean follow-up was 13.5 months. CONCLUSIONS: In these patients bilateral modified Harada-Ito procedures successfully reduced torsion and decreased symptoms. Many patients require subsequent surgery to improve other aspects of their motility problem.

The role of telemedicine in the assessment of strabismus.

Thirty patients with strabismus were seen face to face by an ophthalmologist and an orthoptist. The patients were then presented by the same orthoptist to a second ophthalmologist via a telemedicine link. Twenty-six patients were seen using a bandwidth of 384 kbit/s and four using 128 kbit/s. There was agreement between the two ophthalmologists about diagnosis and management in 24 cases, partial agreement in one and no agreement in five (17%). Manifest strabismus was safely diagnosed and managed using telemedicine at 128 kbit/s, although 384 kbit/s was preferred because it obviated the need for repeated examination. Latent strabismus and micro-movements were difficult to diagnose using telemedicine even at 384 kbit/s. Young patients who are unable to sit still would not be suitable for strabismus assessment via telemedicine.