Effects of motor cortical and peripheral axonal hyperexcitability on survival in amyotrophic lateral sclerosis.

BACKGROUND: Increased 'cortical' and 'peripheral' excitability are reportedly associated with shorter survival in amyotrophic lateral sclerosis (ALS) patients, suggesting that hyperexcitability contributes to motor neuron death. However, whether upper or lower motor function has a greater impact on survival is unclear. We aimed to investigate the component that strongly impacts the prognosis of ALS. METHODS: A total of 103 consecutive patients with ALS who underwent cortical (threshold tracking transcranial magnetic stimulation (TMS)) and motor nerve excitability tests were included. Motor cortical excitability was evaluated using short-interval intracortical inhibition (SICI) during TMS. Motor axonal excitability was assessed using the strength-duration time constant (SDTC). Survival time was defined as the time from examination to death or tracheostomy. RESULTS: Compared with healthy subjects, patients with ALS had lower SICI and longer SDTC (p<0.05), indicating increased excitability of cortical motor neurons and motor axons. According to the SICI and SDTC findings, patients were divided into the following four groups: 'cortical high and peripheral high (high-high)', 'high-low', 'low-high' and 'low-low' groups. In Kaplan-Meier curves, the 'high-high' and 'low-high' groups showed significantly shorter survival than the other groups. Multivariate analysis revealed that increased cortical (HR=5.3, p<0.05) and peripheral (HR=20.0, p<0.001) excitability were significantly associated with shorter survival. CONCLUSIONS: In patients with ALS, both motor cortical and peripheral hyperexcitability independently affected survival time, with peripheral hyperexcitability having a greater impact on shorter survival. The modulation of neuronal/axonal excitability is a potential therapeutic target for ALS.

Impact of antiallergy agents on CPAP therapy and sleep quality with spring pollinosis in Japanese.

PURPOSE: Allergic rhinitis (AR) is associated with obstructive sleep apnea (OSA) and nasal obstruction causes decreased adherence to continuous positive airway pressure (CPAP). The purpose is to evaluate the effects of antiallergic agents on CPAP adherence and sleep quality. METHODS: A longitudinal study was made of patients who use CPAP for OSA and treated with antiallergy agents for spring pollinosis. We compared the Pittsburgh Sleep Quality Index (PSQI), Epworth Sleepiness Scale (ESS), nasal symptoms scores (NSS), and data from CPAP before and after treatment. Then, we classified the subjects into two groups based on the baseline PSQI score: one group without a decreased sleep quality (PSQI < 6) and the other group with decreased sleep quality (PSQI ≥ 6). RESULTS: Of 28 subjects enrolled, 13 had good sleep quality and 15 had poor sleep quality. PSQI showed significant improvements after medication (p = 0.046). ESS showed no significant differences after AR medication (p = 0.565). Significant improvement was observed after the prescription of antiallergy agents in all items of NSS (sneezing, p < 0.05; rhinorrhea, p < 0.01; nasal obstruction, p < 0.01; QOL, p < 0.01). The percentage of days with CPAP use more than 4 h increased significantly after the administration of rhinitis medication (p = 0.022). In the intragroup comparisons of PSQI ≥ 6 group, PSQI decreased significantly (p < 0.05). For the NSS in intragroup comparisons of PSQI ≥ 6 group, all parameters showed significant improvement (sneezing, p = 0.016; rhinorrhea, p = 0.005; nasal obstruction, p < 0.005; QOL, p < 0.005). CONCLUSION: The use of antiallergy agents can improve CPAP adherence and sleep quality in patients with OSA on CPAP.

Relationship between motor cortical and peripheral axonal hyperexcitability in amyotrophic lateral sclerosis.

BACKGROUND: Previous studies have shown that patients with amyotrophic lateral sclerosis (ALS) have hyperexcitability in both the motor cortex and peripheral motor axons, but the relationship between central and peripheral excitability has not been fully disclosed. METHODS: Threshold tracking transcranial magnetic stimulation (TMS) and motor nerve excitability testing were prospectively performed in 53 patients with ALS and 50 healthy subjects, and their relations to compound muscle action potential (CMAP) amplitude and revised ALS Functional Rating Scale were cross-sectionally analysed. RESULTS: Compared with controls, patients with ALS showed both cortical and peripheral hyperexcitability; TMS showed reduced short-interval intracortical inhibition (interstimulus interval 1-7 ms) (p<0.001) and shortened silent period (p<0.05), and median nerve excitability testing revealed greater changes in depolarising threshold electrotonus (TEd) and greater superexcitability (p<0.0001, both), suggesting reduced axonal potassium currents. Significant correlations between cortical and peripheral excitability indices were not found. Greater changes in TEd (90-100 ms) (R=-0.33, p=0.03) and superexcitability (R=0.36, p=0.01) were associated with smaller amplitude of CMAP, whereas cortical excitability indices had no correlation with CMAP amplitude. More rapid motor functional decline was associated with only greater TEd (90-100 ms) (ß=0.46, p=0.001). CONCLUSIONS: Our results suggest that in ALS, cortical excitability is continuously high regardless of the extent of the peripheral burden, but peripheral hyperexcitability is associated with the extent of the peripheral burden and disease evolution speed. Alterations of ion channel function may play an important role in ALS pathophysiology.

Blockade of endoplasmic reticulum stress-induced cell death by Ureaplasma parvum vacuolating factor.

Previously, we found that Ureaplasma parvum internalised into HeLa cells and cytosolic accumulation of galectin-3. U. parvum induced the host cellular membrane damage and survived there. Here, we conducted vesicular trafficking inhibitory screening in yeast to identify U. parvum vacuolating factor (UpVF). U. parvum triggered endoplasmic reticulum (ER) stress and upregulated the unfolded protein response-related factors, including BiP, P-eIF2 and IRE1 in the host cells, but it blocked the induction of the downstream apoptotic factors. MicroRNA library screening of U. parvum-infected cells and UpVF-transfected cells identified miR-211 and miR-214 as the negative regulators of the apoptotic cascade under ER stress. Transient expression of UpVF induced HeLa cell death with intracellular vacuolization; however, some stable UpVF transformant survived. U. parvum-infected cervical cell lines showed resistance to actinomycin D, and UpVF stable transformant cell lines exhibited resistance to X-ray irradiation, as well as cisplatin and paclitaxel. UpVF expressing cervical cancer xenografts in nude mice also acquired resistance to cisplatin and paclitaxel. A mycoplasma expression vector based on Mycoplasma mycoides, Syn-MBA (multiple banded antigen)-UpVF, reduced HeLa cell survival compared with that of Syn-MBA after 72 hr of infection. These findings together suggest novel mechanisms for Ureaplasma infection and the possible implications for cervical cancer malignancy. TAKE AWAYS: • Ureaplasmal novel virulence factor, UpVF, was identified. • UpVF triggered ER stress but suppressed apoptotic cascade via miR-211 and -214. • UpVF conferred resistance to anticancer treatments both in vivo and in vitro. • Dual expression of MBA and UpVF in JCVI-syn3B showed host cell damage.

Different patterns of sensory nerve involvement in chronic inflammatory demyelinating polyneuropathy subtypes.

INTRODUCTION/AIMS: Among subtypes of chronic inflammatory demyelinating polyneuropathy (CIDP), different immune pathophysiologies have been proposed. In this study, sensory nerve conduction studies were compared among clinical subtypes to attempt to better understand the underlying pathophysiology. METHODS: A total of 138 patients with CIDP was classified into clinical subtypes: typical CIDP (N = 68), multifocal CIDP (N = 27), or other (N = 2). Patients with immunoglobulin M (IgM) neuropathy anti-myelin-associated glycoprotein neuropathy (MAG; N = 19) were also included as disease controls. Sensory nerve action potentials (SNAPs) were recorded in the median, ulnar, and superficial radial and sural nerves. RESULTS: SNAP amplitudes (P < .05) and conduction velocities (P < .01) in the median nerve and conduction velocities (P < .05) in the ulnar nerve were lower in typical CIDP than in multifocal CIDP, whereas those in the radial and sural nerves were comparable in each group. Low median and normal sural SNAP amplitudes were more common in typical CIDP (P < .005) than in multifocal CIDP, suggesting predominant involvement at terminal portions of the nerves. DISCUSSION: Terminal portions of sensory nerves are preferentially affected in typical CIDP compared with multifocal CIDP. These findings might be partially explained by the hypothesis of antibody-mediated demyelination in typical CIDP at the regions where the blood-nerve barrier is anatomically deficient, whereas multifocal CIDP predominantly affects the nerve trunks, largely due to cell-mediated demyelination, with disruption of the blood-nerve barrier.

Fasciculation intensity and limb dominance in amyotrophic lateral sclerosis: a muscle ultrasonographic study.

BACKGROUND AND PURPOSE: Muscle ultrasonography has been increasingly recognized as a useful tool for detection of fasciculations. Separately, concordance between dominant hand and onset side has been reported in amyotrophic lateral sclerosis (ALS). The aim of this study was to reveal the distribution of fasciculations in the whole body, focusing on handedness. METHODS: In 106 consecutive patients with ALS, muscle ultrasonography was systematically performed in 11 muscles (the tongue, and bilateral biceps brachii, 1st dorsal interosseous [FDI], T10-paraspinalis, vastus lateralis and tibialis anterior muscles). The fasciculation intensity was scored from 0 to 3 for each muscle. RESULTS: Fasciculations were more frequently found in the limb muscles than the tongue and paraspinalis. Side and handedness analyses revealed that fasciculation intensity in FDI was significantly more prominent on the right (median [inter-quartile range] 2 [0 - 3]) than left (1.5 [0 - 3]; p = 0.016), and in the dominant hand (2 [1 - 3]) than non-dominant side (1.5 [0 - 3]; p = 0.025). The differences were greater in patients with upper limb onset. There were no side differences in the lower limb muscles. Multivariate analyses showed that male patients had more frequent fasciculations in the dominant FDI (ß = 0.22, p < 0.05). CONCLUSION: More intensive fasciculations are present in the FDI in the dominant hand and gender might be associated with fasciculation intensities. This distribution pattern of fasciculations might be associated with pathogenesis of ALS.

Effect of racial background on motor cortical function as measured by threshold tracking transcranial magnetic stimulation.

A previous study using traditional paired-pulse TMS methods (amplitude-tracking) has reported differences in resting motor threshold (RMT) and short-interval intracortical inhibition (SICI) between healthy subjects of Caucasian and Han Chinese backgrounds, probably due to differences in the skull shape. The amplitude-tracking method delivers stimuli with constant intensity and causes substantial variabilities in motor-evoked potential amplitudes. To overcome this variability, threshold tracking transcranial magnetic stimulation (TT-TMS) has been developed. The present study aimed to investigate whether racial differences in motor cortical function exist, using TT-TMS. A total of 83 healthy volunteers (30 Caucasians, 25 Han Chinese, and 28 Japanese) were included in the present series. In TT-TMS and nerve conduction studies, electrodes were placed on the dominant limb, with measures recorded from the abductor pollicis brevis muscle. Stimulations were delivered with a circular coil, directly above the primary motor cortex. There were no significant differences at all the SICI intervals between races. Similarly, there were no significant differences in other measures of excitability including mean RMT, intracortical facilitation, and cortical silent period. Contrary to traditional amplitude-tracking TMS, motor cortical excitability and thereby motor cortical function is minimally influenced by racial differences when measured by TT-TMS. Recent studies have disclosed that SICI measured by TT-TMS differentiates amyotrophic lateral sclerosis (ALS) from ALS mimic disorders, with high sensitivity and specificity, in Caucasians. This study suggested that TT-TMS can be applied for the ALS diagnosis in Asian patients, as well as Caucasians.NEW & NOTEWORTHY Threshold tracking transcranial magnetic stimulation (TT-TMS) was applied for Caucasians, Han Chinese, and Japanese. No significant differences were found in TMS excitability indexes among races. Recent studies have disclosed that TT-TMS indexes differentiate amyotrophic lateral sclerosis (ALS) from ALS mimic disorders, with high sensitivity and specificity, in Caucasians. This study suggested that TT-TMS can be applied for the ALS diagnosis in Asian patients, as well as Caucasians.

Dispersion of mean consecutive differences in single-fiber electromyography increases diagnostic sensitivity for myasthenia gravis.

INTRODUCTION: In this study we aimed to investigate the dispersion of mean consecutive difference (MCD) of concentric needle jitter studies of patients with myasthenia gravis (MG) and its effect on diagnostic sensitivity for MG. METHODS: One hundred fifty-three patients, including 76 patients with MG and 77 controls with possible MG who later received another diagnosis, underwent stimulated concentric needle jitter studies of the frontalis muscle. MCD mean, standard deviation (SD), and coefficient of variation (CV) were calculated. Diagnostic sensitivity and specificity were determined using receiver operating characteristic (ROC) analyses. RESULTS: MG patients showed a significantly greater MCD mean (MG: control, 26.3 µs; 13.5 µs [median]; P < .0001), MCD SD (MG: control, 12.8 µs; 5.1 µs [median]; P < .0001), and MCD CV (MG: control, 46.1; 37.5 [median]; P < .001) than those without MG. An ROC curve of SD showed a large area under the curve (0.88), and a cut-off value of 7.2 µs, which was calculated by maximum Youden index, exhibited high diagnostic sensitivity (86%) for MG. Combined MCD mean, outliers, and SD criteria showed higher sensitivity (88%) than conventional criteria alone (82%), at the expense of lower specificity. Five MG patients with normal MCD mean and abnormal MCD SD had only ocular symptoms. DISCUSSION: The dispersion of MCD as measured by MCD SD greater than 7.2 µs is significantly increased in patients with MG and may be a useful measure of abnormal jitter in the diagnosis of MG, especially for identifying patients with mild disease.

Clinical, polysomnographic, and cephalometric features of obstructive sleep apnea with AHI over 100.

PURPOSE: Obstructive sleep apnea (OSA) is induced by a sleep-related collapse of the upper airway in association with multiple factors. The severity of OSA is determined by the apnea-hypopnea index (AHI). Although obesity and sex differences are common factors in OSA, the level of the AHI varies to the same degree according to the age and sex and degree of obesity. However, only a few studies have evaluated AHI over 100/h, those reports did not describe why they set the AHI cutoff at 100/h. The purpose of this study was to elucidate the pathogenesis of "very" severe OSA, defined as having an AHI > 100/h. METHODS: AHI > 100/h was set as very severe OSA (VS-OSA) in this study. As controls, moderate to severe OSA patients, matched with VS-OSA for age, sex, and body mass index (BMI), were enrolled. The findings of polysomnography and cephalography between VS-OSA and controls were compared. RESULTS: Eleven patients in the VS-OSA group (mean AHI 110.2/h) and 22 patients in the control group (mean AHI 41.6/h) were compared (mean age 50.2 vs 50.6, male:female 5:6 vs 10:12, mean BMI 35.4 kg/m2 vs 34.5 kg/m2). There were no significant differences in the clinical characteristics. In the polysomnographic parameters, the VS-OSA group showed apnea predominance, the mean percutaneous oxygen saturation (SO2) was significantly lower in all sleep stages, and the minimum SO2 was significantly lower (49.0% vs 77.5%, p = 0.002). A similar apnea duration and rather shorter hypopnea duration were shown. The time of apnea-to-arousal was significantly earlier (- 0.1 s vs 0.9 s, p = 0.003). Lung-to-finger circulation time showed no differences. The cephalometric findings showed no significant differences. CONCLUSIONS: VS-OSA patients were more likely to have apnea predominance, desaturation when sleeping despite a similar apnea duration, and rather shorter hypopnea duration, and arousals were evoked significantly earlier.

Split hand and motor axonal hyperexcitability in spinal and bulbar muscular atrophy.

OBJECTIVE: The 'split hand' sign refers to preferential wasting of the thenar and first dorsal interosseous muscles with relatively sparing of the hypothenar muscles in amyotrophic lateral sclerosis (ALS) and both cortical and spinal/peripheral excitotoxic mechanisms have been proposed. We aimed to study split hand and axonal excitability in spinal and bulbar muscular atrophy (SBMA) in which cortical motor neurons are intact. METHODS: In 35 patients with genetically confirmed SBMA, 55 with ALS, 158 with other neuromuscular diseases and 90 normal controls; split hand was strictly determined by amplitudes of compound muscle action potentials. Nerve excitability testing of median motor axons was performed in 35 SBMA and 55 patients with ALS and 45 normal controls. RESULTS: Split hand was as frequently found for patients with SBMA (57%) and ALS (62%), compared with disease (20%) and normal (0%) controls. Excitability testing showed that in both SBMA and ALS, strength-duration time constant was longer, and threshold changes in depolarising threshold electrotonus and superexcitability in the recovery cycle were greater than in normal controls (p<0.01). CONCLUSIONS: Split hand is not specific to ALS and can be caused by the peripheral mechanism alone in SBMA, whereas the effect of upper motor neuron lesion cannot be excluded in ALS. Our results also suggest that SBMA and ALS share common axonal excitability changes; increased nodal persistent sodium and reduced potassium currents that may accelerate motor neuronal death and differently affect axons-innervating different muscles. Ion channel modulators could be a therapeutic option for both SBMA and ALS.

Membrane property changes in most distal motor axons in chronic inflammatory demyelinating polyneuropathy.

INTRODUCTION: Distal nerve terminals, where the blood-nerve barrier is anatomically deficient, are preferentially affected in immune-mediated neuropathies. Excitability alterations near the motor nerve terminals may be more prominent than the nerve trunk in typical chronic inflammatory demyelinating polyneuropathy (CIDP). METHODS: In 20 patients with typical CIDP, motor nerve excitability testing was performed at the motor point and wrist of the ulnar nerve, and results were compared with those in 20 healthy persons. RESULTS: Chronic inflammatory demyelinating polyneuropathy patients showed greater threshold changes in hyperpolarizing threshold electrotonus at the motor point (P < .05) but not at the wrist. Strength-duration time constant did not show significant differences between CIDP and controls at both sites. DISCUSSION: Axonal property changes in CIDP are more prominent in distal portions of axons compared with the nerve trunk, presumably due to salient demyelination near the distal nerve terminals. Motor point excitability measurements could elucidate underlying pathophysiology in immune-mediated neuropathies.

Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling.

The availability of genetically tractable organisms with simple genomes is critical for the rapid, systems-level understanding of basic biological processes. Mycoplasma bacteria, with the smallest known genomes among free-living cellular organisms, are ideal models for this purpose, but the natural versions of these cells have genome complexities still too great to offer a comprehensive view of a fundamental life form. Here we describe an efficient method for reducing genomes from these organisms by identifying individually deletable regions using transposon mutagenesis and progressively clustering deleted genomic segments using meiotic recombination between the bacterial genomes harbored in yeast. Mycoplasmal genomes subjected to this process and transplanted into recipient cells yielded two mycoplasma strains. The first simultaneously lacked eight singly deletable regions of the genome, representing a total of 91 genes and ∼ 10% of the original genome. The second strain lacked seven of the eight regions, representing 84 genes. Growth assay data revealed an absence of genetic interactions among the 91 genes under tested conditions. Despite predicted effects of the deletions on sugar metabolism and the proteome, growth rates were unaffected by the gene deletions in the seven-deletion strain. These results support the feasibility of using single-gene disruption data to design and construct viable genomes lacking multiple genes, paving the way toward genome minimization. The progressive clustering method is expected to be effective for the reorganization of any mega-sized DNA molecules cloned in yeast, facilitating the construction of designer genomes in microbes as well as genomic fragments for genetic engineering of higher eukaryotes.

Direct transfer of whole genomes from bacteria to yeast.

Transfer of genomes into yeast facilitates genome engineering for genetically intractable organisms, but this process has been hampered by the need for cumbersome isolation of intact genomes before transfer. Here we demonstrate direct cell-to-cell transfer of bacterial genomes as large as 1.8 megabases (Mb) into yeast under conditions that promote cell fusion. Moreover, we discovered that removal of restriction endonucleases from donor bacteria resulted in the enhancement of genome transfer.

Strategies for cloning and manipulating natural and synthetic chromosomes.

Advances in synthetic biology methods to assemble and edit DNA are enabling genome engineering at a previously impracticable scale and scope. The synthesis of the Mycoplasma mycoides genome followed by its transplantation to convert a related cell into M. mycoides has transformed strain engineering. This approach exemplifies the combination of newly emerging chromosome-scale genome editing strategies that can be defined in three main steps: (1) chromosome acquisition into a microbial engineering platform, (2) alteration and improvement of the acquired chromosome, and (3) installation of the modified chromosome into the original or alternative organism. In this review, we outline recent progress in methods for acquiring chromosomes and chromosome-scale DNA molecules in the workhorse organisms Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae. We present overviews of important genetic strategies and tools for each of the three organisms, point out their respective strengths and weaknesses, and highlight how the host systems can be used in combination to facilitate chromosome assembly or engineering. Finally, we highlight efforts for the installation of the cloned/altered chromosomes or fragments into the target organism and present remaining challenges in expanding this powerful experimental approach to a wider range of target organisms.

Genomic and transcriptomic analyses of colistin-resistant clinical isolates of Klebsiella pneumoniae reveal multiple pathways of resistance.

The emergence of multidrug-resistant (MDR) Klebsiella pneumoniae has resulted in a more frequent reliance on treatment using colistin. However, resistance to colistin (Col(r)) is increasingly reported from clinical settings. The genetic mechanisms that lead to Col(r) in K. pneumoniae are not fully characterized. Using a combination of genome sequencing and transcriptional profiling by RNA sequencing (RNA-Seq) analysis, distinct genetic mechanisms were found among nine Col(r) clinical isolates. Col(r) was related to mutations in three different genes in K. pneumoniae strains, with distinct impacts on gene expression. Upregulation of the pmrH operon encoding 4-amino-4-deoxy-L-arabinose (Ara4N) modification of lipid A was found in all Col(r) strains. Alteration of the mgrB gene was observed in six strains. One strain had a mutation in phoQ. Common among these seven strains was elevated expression of phoPQ and unaltered expression of pmrCAB, which is involved in phosphoethanolamine addition to lipopolysaccharide (LPS). In two strains, separate mutations were found in a previously uncharacterized histidine kinase gene that is part of a two-component regulatory system (TCRS) now designated crrAB. In these strains, expression of pmrCAB, crrAB, and an adjacent glycosyltransferase gene, but not that of phoPQ, was elevated. Complementation with the wild-type allele restored colistin susceptibility in both strains. The crrAB genes are present in most K. pneumoniae genomes, but not in Escherichia coli. Additional upregulated genes in all strains include those involved in cation transport and maintenance of membrane integrity. Because the crrAB genes are present in only some strains, Col(r) mechanisms may be dependent on the genetic background.

Enhanced interference-pattern visibility using multislit optical superposition method for imaging-type two-dimensional Fourier spectroscopy.

A solution is found for the problem of phase cancellation between adjacent bright points in wavefront-division phase-shift interferometry. To this end, a design is proposed that optimizes the visibility of the interference pattern from multiple slits. The method is explained in terms of Fraunhofer diffraction and convolution imaging. Optical simulations verify the technique. The final design can be calculated using a simple equation.

Knocking out multigene redundancies via cycles of sexual assortment and fluorescence selection.

Phenotypes that might otherwise reveal a gene's function can be obscured by genes with overlapping function. This phenomenon is best known within gene families, in which an important shared function may only be revealed by mutating all family members. Here we describe the 'green monster' technology that enables precise deletion of many genes. In this method, a population of deletion strains with each deletion marked by an inducible green fluorescent protein reporter gene, is subjected to repeated rounds of mating, meiosis and flow-cytometric enrichment. This results in the aggregation of multiple deletion loci in single cells. The green monster strategy is potentially applicable to assembling other engineered alterations in any species with sex or alternative means of allelic assortment. To test the technology, we generated a single broadly drug-sensitive strain of Saccharomyces cerevisiae bearing precise deletions of all 16 ATP-binding cassette transporters within clades associated with multidrug resistance.

Cloning and sequencing analysis of whole Spiroplasma genome in yeast.

Cloning and transfer of long-stranded DNA in the size of a bacterial whole genome has become possible by recent advancements in synthetic biology. For the whole genome cloning and whole genome transplantation, bacteria with small genomes have been mainly used, such as mycoplasmas and related species. The key benefits of whole genome cloning include the effective maintenance and preservation of an organism's complete genome within a yeast host, the capability to modify these genome sequences through yeast-based genetic engineering systems, and the subsequent use of these cloned genomes for further experiments. This approach provides a versatile platform for in-depth genomic studies and applications in synthetic biology. Here, we cloned an entire genome of an insect-associated bacterium, Spiroplasma chrysopicola, in yeast. The 1.12 Mbp whole genome was successfully cloned in yeast, and sequences of several clones were confirmed by Illumina sequencing. The cloning efficiency was high, and the clones contained only a few mutations, averaging 1.2 nucleotides per clone with a mutation rate of 4 × 10-6. The cloned genomes could be distributed and used for further research. This study serves as an initial step in the synthetic biology approach to Spiroplasma.