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.

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.

Improved Combinatorial Assembly and Barcode Sequencing for Gene-Sized DNA Constructs.

Synergistic and supportive interactions among genes can be incorporated in engineering biology to enhance and stabilize the performance of biological systems, but combinatorial numerical explosion challenges the analysis of multigene interactions. The incorporation of DNA barcodes to mark genes coupled with next-generation sequencing offers a solution to this challenge. We describe improvements for a key method in this space, CombiGEM, to broaden its application to assembling typical gene-sized DNA fragments and to reduce the cost of sequencing for prevalent small-scale projects. The expanded reach of the method beyond currently targeted small RNA genes promotes the discovery and incorporation of gene synergy in natural and engineered processes such as biocontainment, the production of desired compounds, and previously uncharacterized fundamental biological mechanisms.

Drainage Tube Placement for the Management of Rebleeding After Vascular Embolization in a Patient With Neurofibromatosis Type 1.

Neurofibromatosis type 1 (NF-1), a hereditary disease caused by a mutation of a gene on chromosome 17q11.2, is associated with manifestations in several organs. Although infrequent, vascular abnormalities are a complication of NF-1, and they are the second most common cause of death in patients with NF-1. Repairing the nutrient artery and achieving hemostasis are difficult once the artery has failed, thereby resulting in poor treatment outcomes. Herein, we report a case of a patient with NF-1 who presented with an enormous cervical hematoma caused by bleeding from a branch of the external carotid artery. Vascular embolization was performed initially; however, rebleeding from the embolized site occurred. Following the removal of the hematoma, drainage tube placement was effective in preventing micro-bleeding. Thus, drainage tube placement may be an effective treatment option in patients with rebleeding.

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.

Comparison of different methods for evaluating the relationship between facial nerve and benign parotid tumors.

Objectives: The goals of benign parotid gland tumor resection are complete resection of the lesion and preservation of the facial nerve function. As the facial nerve cannot be directly visualized via imaging modalities, several methods, including the facial nerve line, Utrecht line, retromandibular vein, Stenon duct, and minimum fascia-tumor distance techniques, have been developed to estimate its location. However, there are no reports on their accuracy in determining tumor location. In the present study, we aimed to assess the diagnostic accuracy of these methods based on tumor location. Methods: This retrospective study analyzed medical records and histological reports of 359 patients with various types of benign parotid gland tumors who underwent a parotidectomy between April 2014 and March 2020. The tumor location was subdivided into the following sections: anterior, superior, inferior, and middle. The tumor location was estimated using five methods: facial nerve line, Utrecht line, retromandibular vein, Stenon duct, and minimum fascia-tumor distance. The final diagnosis of superficial or deep lobe tumor was made based on surgical findings. Results: Each method showed a higher accuracy for superficial tumors (was more than 90%) than for deep lobe tumors. In contrast, for deep lobe tumors, the accuracy of diagnosis with the facial nerve line, Utrecht line, and retromandibular vein methods was low, in the 30% range. Among all methods, the Stenon duct method had the highest accuracy in the diagnosis of deep lobe tumors. The SD method was most useful in cases where both the duct and tumors were detected. The minimum fascia-tumor distance method had the second highest diagnostic accuracy (63%); however, for anterior tumors, it tended to provide false negatives. Conclusions: All tested methods were useful in diagnosing superficial lobe tumors; however, they were not helpful in diagnosing deep lobe tumors, especially anterior tumors.

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.

Impact of radiotherapy for head and neck cancer on obstructive sleep apnea: a prospective study.

BACKGROUND: In recent years, a relatively high prevalence of obstructive sleep apnea (OSA) in patients following radiotherapy (RT) for head and neck cancer (HNC) has been reported; however, little is known regarding the impact of RT on sleep disorders and the underlying mechanisms. This aim of this study was to elucidate the pathogenesis of OSA by comparing the clinical and sleep test parameters and magnetic resonance imaging (MRI) findings before and after HNC treatment with radiation. METHODS: This prospective study included patients scheduled for RT with or without chemotherapy or bioradiotherapy for HNC. Patients diagnosed with HNC between May 2017 and August 2020 were consecutively recruited. The results of the sleep tests were analyzed both before and after treatment. The clinical characteristics of the patients and cephalometric and MRI parameters were also measured. RESULTS: First, a total of 32 patients (64.8±11.8 years old; BMI, 22.7±3.6 kg/m2) underwent pre-treatment sleep tests. The prevalence of OSA [apnea hypopnea index (AHI) ≥5] in these patients was 81.3% (26 patients) before treatment, and the mean AHI was 20.8±19.0 events/hr. Next, 21 patients performed a sleep test both before and after treatment. Regarding subjective symptoms, there were no significant differences in the Epworth Sleepiness Scale (ESS) (P=0.142) or Pittsburgh Sleep Quality Index (PSQI) (P=0.935) after treatment; however, the BMI and neck circumference significantly decreased after treatment (P<0.0001 and P=0.0001, respectively). The incidence of OSA in these patients was 81.0% (17 patients) before treatment and 85.7% (19 patients) after treatment (P=1.0). Overall, the AHI was not significantly different, changing only from 14.5 to 14.9 after treatment (P=0.147). The MRI parameters showed that the retroglossal pharyngeal area increased significantly after treatment (P=0.007). CONCLUSIONS: This study found that the prevalence of OSA before and after RT for HNC was higher than that in the normal population, despite a significant decrease in BMI and increase in the retroglossal pharyngeal area after treatment. We suggest that physicians who manage patients with HNC should consider the occurrence of OSA before and after treatment.

Neuronal Hyperexcitability and Free Radical Toxicity in Amyotrophic Lateral Sclerosis: Established and Future Targets.

Amyotrophic lateral sclerosis (ALS) is a devastating disease with evidence of degeneration involving upper and lower motor neuron compartments of the nervous system. Presently, two drugs, riluzole and edaravone, have been established as being useful in slowing disease progression in ALS. Riluzole possesses anti-glutamatergic properties, while edaravone eliminates free radicals (FRs). Glutamate is the excitatory neurotransmitter in the brain and spinal cord and binds to several inotropic receptors. Excessive activation of these receptors generates FRs, inducing neurodegeneration via damage to intracellular organelles and upregulation of proinflammatory mediators. FRs bind to intracellular structures, leading to cellular impairment that contributes to neurodegeneration. As such, excitotoxicity and FR toxicities have been considered as key pathophysiological mechanisms that contribute to the cascade of degeneration that envelopes neurons in ALS. Recent advanced technologies, including neurophysiological, imaging, pathological and biochemical techniques, have concurrently identified evidence of increased excitability in ALS. This review focuses on the relationship between FRs and excitotoxicity in motor neuronal degeneration in ALS and introduces concepts linked to increased excitability across both compartments of the human nervous system. Within this cellular framework, future strategies to promote therapeutic development in ALS, from the perspective of neuronal excitability and function, will be critically appraised.

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.

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.

Fatigue and activity-dependent conduction block in neuromuscular disorders.

Objective: Fatigue is a major disabling problem in patients with neuromuscular disorders. Both nerve demyelination and increased axonal branching associated with collateral sprouting reduce the safety factor for impulse transmission and could cause activity-dependent hyperpolarization and conduction block during voluntary contraction, and thus fatigue. This study aimed to investigate whether activity-dependent conduction block is associated with fatigue in demyelinating neuropathies and lower motor neuron disorders. Methods: This study included 31 patients (17 with chronic inflammatory demyelinating polyneuropathy [CIDP] and 14 with spinal and bulbar muscular atrophy [SBMA]). Sixteen healthy subjects served as normal controls. Fatigue was assessed using the Fatigue Scale for Motor and Cognitive Functions (FSMC). Compound muscle action potential (CMAP) recording and nerve excitability testing after median nerve stimulation in the wrist were performed before and after maximal voluntary contraction of the abductor pollicis brevis for 1 min. Results: Patients with CIDP/SBMA had prominent fatigue with higher FSMC motor scores (P < 0.0001) than normal controls. After voluntary contractions, CMAP amplitudes decreased significantly in four of the 17 patients with CIDP and one of the 14 patients with SBMA. The reduction in CMAP amplitude was associated with the fatigue score in the motor but not in the cognitive domain. After voluntary contraction, excitability testing showed axonal hyperpolarization in the normal and CIDP/SBMA groups. Conclusions: In CIDP or SBMA, fatigue is caused by voluntary contraction-induced membrane hyperpolarization and conduction block, presumably due to the critically lowered safety factor due to demyelination or increased axonal branching. Significance: Peripheral fatigue can be objectively assessed using CMAP amplitudes and nerve excitability testing.

Adaptive laboratory evolution in S. cerevisiae highlights role of transcription factors in fungal xenobiotic resistance.

In vitro evolution and whole genome analysis were used to comprehensively identify the genetic determinants of chemical resistance in Saccharomyces cerevisiae. Sequence analysis identified many genes contributing to the resistance phenotype as well as numerous amino acids in potential targets that may play a role in compound binding. Our work shows that compound-target pairs can be conserved across multiple species. The set of 25 most frequently mutated genes was enriched for transcription factors, and for almost 25 percent of the compounds, resistance was mediated by one of 100 independently derived, gain-of-function SNVs found in a 170 amino acid domain in the two Zn2C6 transcription factors YRR1 and YRM1 (p < 1 × 10-100). This remarkable enrichment for transcription factors as drug resistance genes highlights their important role in the evolution of antifungal xenobiotic resistance and underscores the challenge to develop antifungal treatments that maintain potency.

Severe obstructive sleep apnea after concurrent chemoradiotherapy for laryngeal and hypopharyngeal cancer managed by CPAP.

Concurrent chemoradiotherapy (CCRT) is one of the most promising treatments for advanced head and neck cancer (HNC). On the other hand, CCRT may induce severe edema in laryngo-pharyngeal structures in association with radiation. This is a report of a 66-year-old man with severe obstructive sleep apnea (OSA) associated with edema in laryngo-hypopharynx after CCRT for advanced laryngeal and hypopharyngeal cancer. Tracheostomy was avoided and OSA was controlled by continuous positive airway pressure (CPAP). Subjective symptoms of sleepiness were improved. Though laryngeal edema appeared during the course of CCRT in this case, OSA was not evaluated until snoring had been pointed out and he complained of sleepiness. CCRT for laryngeal and hypopharyngeal cancer have a risk of occurrence of OSA due to irreversible mucous edema in the upper airway. Patients for whom CCRT is planned should be informed about the occurrence of OSA before the treatment because symptoms associated with OSA can negatively impact not only the daytime quality of life but also increase the risk of cardiovascular events. The OSA treatment for post CCRT would be expected to have a positive impact on not only cardiovascular and metabolic systems but also on the cancer treatment survival rate.

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.

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.

Biotechnology for secure biocontainment designs in an emerging bioeconomy.

Genetically modified organisms (GMOs) have emerged as an integral component of a sustainable bioeconomy, with an array of applications in agriculture, bioenergy, and biomedicine. However, the rapid development of GMOs and associated synthetic biology approaches raises a number of biosecurity concerns related to environmental escape of GMOs, detection thereof, and impact upon native ecosystems. A myriad of genetic safeguards have been deployed in diverse microbial hosts, ranging from classical auxotrophies to global genome recoding. However, to realize the full potential of microbes as biocatalytic platforms in the bioeconomy, a deeper understanding of the fundamental principles governing microbial responsiveness to biocontainment constraints, and interactivity of GMOs with the environment, is required. Herein, we review recent analytical biotechnological advances and strategies to assess biocontainment and microbial bioproductivity, as well as opportunities for predictive systems biodesigns towards securing a viable bioeconomy.

Corrigendum to "A Case of Epistaxis as the First Sign of Acute Idiopathic Thrombocytopenic Purpura".

[This corrects the article DOI: 10.1155/2021/6612939.].