The characteristics of autolysins associated with cell separation in Bacillus subtilis.

The peptidoglycan hydrolases responsible for the cell separation of Bacillus subtilis cells are collectively referred to as autolysins. However, the role of each autolysin in the cell separation of B. subtilis is not fully understood. In this study, we constructed a series of cell separation-associated autolysin deficient strains and strains overexpressing the transcription factors SlrR and SinR, and the morphological changes of these strains in liquid culture were observed. The results showed that the absence of D,L-endopeptidases CwlS and LytF only increased the cell chain length in the early exponential phase. The absence of D,L-endopeptidase LytE or N-acetylmuramyl-L-alanine amidase LytC can cause cells to form chains throughout the growth of B. subtilis, although the cell chain length was significantly shortened during the stationary phase. However, the absence of peptidoglycan N-acetylglucosaminidase LytD only caused minor defect in cell separation. Therefore, we concluded that LytE and LytC were the major autolysins that ensure the timely separation of B. subtilis daughter cells, whereas CwlS, LytF, and LytD were the minor autolysins. In addition, overexpression of the transcription factors SinR and SlrR in the cwlS lytF lytC lytE mutant enabled B. subtilis cells to form ultra-long chains in the vegetative phase, and its biomass level was basically the same as that of the wild type. This led to the conclusion that besides inhibiting the expression of lytC and lytF, the SinR-SlrR complex also has other potential mechanisms to inhibit cell separation.IMPORTANCEIn this study, the effects of CwlS, LytC, LytD, LytF, LytE, and SinR-SlrR complex on the cell separation of Bacillus subtilis at different growth phases were studied, and an ultra-long-chained B. subtilis strain was constructed. In microbial fermentation, due to its large cell size, this ultra-long-chained B. subtilis strain may be more likely to be precipitated or intercepted during the removal of bacterial process with centrifugation and membrane filtration as the main methods, which is crucial to improve the purity of the product.

Metal-organic polymer enables efficient organic photoelectrochemical transistor biosensing.

The field of organic photoelectrochemical transistor (OPECT) is newly emerged, with increasing efforts attempting to utilize its properties in biological sensing. Advanced materials with new physicochemical properties have proven important to this end. Herein, we report a metal-organic polymers-gated OPECT biosensing exemplified by CuⅠ-arylacetylide polymers (CuAs)-modulated poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) channel. Both the photoelectrochemical properties and gating capability of CuAs are explored and optimized for high-efficacy photogating. Morever, based on its inherent structure, the specific reaction between CuAs and sulfur ions (S2-) is revealed and S2--mediated microRNA-21 detection is realized by linking with nucleic acid amplification and alkaline phosphatase catalytic chemistry. This work introduces metal-organic polymers as gating materials for OPECT biosensing.

Force-Directed Graph Layouts Revisited: A New Force Based on the T-Distribution.

In this paper, we propose the t-FDP model, a force-directed placement method based on a novel bounded short-range force (t-force) defined by Student's t-distribution. Our formulation is flexible, exerts limited repulsive forces for nearby nodes and can be adapted separately in its short- and long-range effects. Using such forces in force-directed graph layouts yields better neighborhood preservation than current methods, while maintaining low stress errors. Our efficient implementation using a Fast Fourier Transform is one order of magnitude faster than state-of-the-art methods and two orders faster on the GPU, enabling us to perform parameter tuning by globally and locally adjusting the t-force in real-time for complex graphs. We demonstrate the quality of our approach by numerical evaluation against state-of-the-art approaches and extensions for interactive exploration.

Identification of novel FHL1 mutations associated with X-linked scapuloperoneal myopathy in unrelated Chinese patients.

Mutations in the FHL1 gene can be associated with a variety of X-linked myopathies and cardiomyopathies, among which X-linked dominant scapuloperoneal myopathy is a rare phenotype. We collected the clinical data of two unrelated Chinese patients with X-linked scapuloperoneal myopathy and analyzed their clinical, pathological, muscle imaging, and genetic features. Both patients were characterized by scapular winging, bilateral Achilles tendon contractures, and weakness in shoulder-girdle and peroneal muscles. Muscle biopsy revealed myopathic changes, and no reducing bodies were found. Muscle magnetic resonance imaging was dominated by fatty infiltration, with minor edema-like findings. Genetic analysis revealed two novel mutations in the FHL1 gene: c.380T > C (p.F127S) and c.802C > T (p.Q268*), which were located in the LIM2 domain and the C-terminal sequence, respectively. To our knowledge, this is the first report of X-linked scapuloperoneal myopathy in the Chinese population. Our findings broadened the genetic and ethnic spectrum of FHL1-related disorders and proposed to look for variants in the FHL1 gene when scapuloperoneal myopathy is observed in the clinical work.

Alkaline Phosphatase-Mediated Bioetching of CoOOH/BiVO4 for Signal-On Organic Photoelectrochemical Transistor Bioanalysis.

Organic photoelectrochemical transistor (OPECT) bioanalytics has recently appeared as a promising route for biological measurements, which has major implications in both next-generation photoelectrochemical (PEC) bioanalysis and futuristic biorelated implementations. Via biological dissociation of materials, bioetching is a useful technique for bio-manufacturing and bioanalysis. The intersection of these two domains is expected to be a possible way to achieve innovative OPECT bioanalytics. Herein, we validate such a possibility, which is exemplified by alkaline phosphatase (ALP)-mediated bioetching of a CoOOH/BiVO4 gate for a signal-on OPECT immunoassay of human immunoglobulin G (HIgG) as the model target. Specifically, target-dependent bioetching of the upper CoOOH layer could result into an enhanced electrolyte contact and light accessibility to BiVO4, leading to the modulated response of the polymeric poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) channel that could be monitored by the channel current. The introduced biosensor achieves sensitive detection of HIgG with high selectivity and sensitivity. This work features bioetching-enabled high-efficacy OPECT bioanalysis and is anticipated to serve as a generic protocol, considering the diverse bioetching routes.

Biological modulating organic photoelectrochemical transistor through in situ enzymatic engineering of photoactive gate for sensitive detection of serum alkaline phosphatase.

Innovative optoelectronics are expected to play more important role in clinical diagnosis. In this study, on the basis of sensitive gating effect by in situ enzymatic functionalization of semiconductors, a novel organic photoelectrochemical transistor (OPECT) detection of serum alkaline phosphatase (ALP) level was demonstrated. Specifically, the OPECT detection operates upon the ALP-catalyzed hydrolysis of sodium thiophosphate to yield hydrogen sulfide (H2S), which could in situ generate CdS on the TiO2 electrode in the presence of Cd2+ cations. Correlated to the ALP level, the CdS directly formed on and interfacing with the TiO2 could sensitively gating the poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) channel, allowing unique optoelectronic detection of serum ALP level with a linear range from 0.005 to 15 U L-1 and a detection limit corresponding to 0.0012 U L-1 (S/N = 3). This study offers not only an optoelectronic method for detection of serum ALP level, but also a perspective for unique OPECT gating and application. Moreover, the general catalytic abilities of enzymes to produce functional species and their rich interactions with various gate substrates further provide great space for futuristic OPECT detection in enzyme-associated diseases.

Semiconducting metal-organic framework derivatives-gated organic photoelectrochemical transistor immunoassay.

Metal-organic framework (MOF) derivatives with unique physicochemical and electronic properties have seen a tremendous growth in diverse applications. Organic optobioelectronics have long been pursued in modern electronics for next-generation bio-relevant implementations. The intersection of these two disciplines could be an appealing way to pursue better performance of materials and devices. Herein this work reports the exploration of MOF derivatives and its ionic modulation for gating organic photoelectrochemical transistor (OPECT) biosensing. In the representative system of poly(ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) gated by zeolitic-imidazolate-framework (ZIF)-8-derived CdxZn1-xS, a high current gain could be achieved at zero gate bias. In connection to a CuO nanoparticle-labeled sandwich immunoassay, acidolysis-triggered Cu2+-induced ionic modulation of the system results into a good performance toward human IgG with a low limit of detection of 0.003 pg/mL. This work features the MOF derivative-gated organic electronics and is expected to inspire more interest to explore various MOF derivative electronics with unknown possibilities, considering the diversity of MOF derivatives.

Tuning the Surface Molecular Charge of Organic Photoelectrochemical Transistors with Significantly Improved Signal Resolution: A General Strategy toward Sensitive Bioanalysis.

Nature makes use of molecular charges to operate specific biological synthesis and reactions. Targeting advanced opto-bioelectronic sensors, organic photoelectrochemical transistors (OPECTs), taking advantage of the light fuel substituting an external gate potential, is now debuting and expected to serve as a universal platform for studying the rich light-biomatter interplay for new bioanalytics. Given the ubiquity of charged biomolecules in nature, molecular charge manipulation should underpin a generic route for innovative OPECT regulation and operation, which nevertheless has remained unachieved. Herein, this work manifests the biological tuning of surface charge toward the OPECT biosensor, which was exemplified by a light-sensitive CdS quantum dot (QD) gate electrode interfaced by a smart DNA superstructure with adenosine triphosphate (ATP) responsiveness. Highly negative-charged supramolecular DNA concatemers were self-assembled via sequential hybridization, and the ATP-triggered disassembly of the DNA concatemers would cause a tandem change of the effective gate voltage and transfer characteristics with significantly improved resolution. The present opto-bioelectronic device translates the events of charged molecules into amplified electrical signals and outlines a generic format for the future exploitation of rich biological tunability and light-biomatter interplay for innovative bioanalytics and beyond.

Construction of a mutant Bacillus subtilis strain for high purity poly-γ-glutamic acid production.

OBJECTIVE: To construct a Bacillus subtilis strain for improved purity of poly-γ-glutamic acid. RESULTS: The construction of strain GH16 was achieved by knocking out five genes encoding extracellular proteins and an operon from Bacillus subtilis G423. We then analyzed the amount of protein impurities in the γ-PGA produced by the resulting strain GH16/pHPG, which decreased from 1.48 to 1.39%. Subsequently the fla-che operon, PBSX, as well as the yrpD, ywoF and yclQ genes were knocked out successively, resulting in the mutant strains GH17, GH18 and GH19. Ultimately, the amount of protein impurities was reduced from 1.48 to 0.83%. In addition, the amount of polysaccharide impurities in the γ-PGA was also decreased from 2.21 to 1.93% after knocking out the epsA-O operon. CONCLUSIONS: The high purity γ-PGA producer was constructed, and the resulting strain was a promising platform for the manufacture of other highly pure extracellular products and secretory proteins.

Comparison of the Unfolded Protein Response in Cellobiose Utilization of Recombinant Angel- and W303-1A-Derived Yeast Expressing ß-Glucosidase.

The unfolded protein response (UPR) is one of the most important protein quality control mechanisms in cells. At least, three factors are predicted to activate the UPR in yeast cells during fermentation. Using UPRE-lacZ as a reporter, we constructed two indicator strains, KZ and WZ, based on Angel-derived K-a and W303-1A strains, respectively, and investigated their UPR response to tunicamycin, ethanol, and acetic acid. Then, four strains carrying plasmids BG-cwp2 and BG were obtained to realize the displaying and secretion of ß-glucosidase, respectively. The results of cellobiose utilization assays indicated interactions between the UPR and the metabolic burden between the strain source, anchoring moiety, oxygen supply, and cellobiose concentration. Meanwhile, as expected, growth (OD600), ß-glucosidase, and ß-galactosidase activities were shown to have a positive inter-relationship, in which the values of the KZ-derived strains were far lower than those of the WZ-derived strains. Additionally, extra metabolic burden by displaying over secreting was also much more serious in strain KZ than in strain WZ. The maximum ethanol titer of the four strains (KZ (BG-cwp2), KZ (BG), WZ (BG-cwp2), and WZ (BG)) in oxygen-limited 10% cellobiose fermentation was 3.173, 5.307, 5.495, and 5.486% (v/v), respectively, and the acetic acid titer ranged from 0.038 to 0.060% (v/v). The corresponding maximum values of the ratio of ß-galactosidase activity to that of the control were 3.30, 5.29, 6.45, and 8.72, respectively. Under aerobic conditions with 2% cellobiose, those values were 3.79, 4.97, 6.99, and 7.67, respectively. A comparison of the results implied that ß-glucosidase expression durably induced the UPR, and the effect of ethanol and acetic acid depended on the titer produced. Further study is necessary to identify ethanol- or acid-specific target gene expression. Taken together, our results indicated that the host strain W303-1A is a better secretory protein producer, and the first step to modify strain K-a for cellulosic ethanol fermentation would be to relieve the bottleneck of UPR capacity. The results of the present study will help to identify candidate host strains and optimize expression and fermentation by quantifying UPR induction.

Clinical implementation of RNA sequencing for Mendelian disease diagnostics.

BACKGROUND: Lack of functional evidence hampers variant interpretation, leaving a large proportion of individuals with a suspected Mendelian disorder without genetic diagnosis after whole genome or whole exome sequencing (WES). Research studies advocate to further sequence transcriptomes to directly and systematically probe gene expression defects. However, collection of additional biopsies and establishment of lab workflows, analytical pipelines, and defined concepts in clinical interpretation of aberrant gene expression are still needed for adopting RNA sequencing (RNA-seq) in routine diagnostics. METHODS: We implemented an automated RNA-seq protocol and a computational workflow with which we analyzed skin fibroblasts of 303 individuals with a suspected mitochondrial disease that previously underwent WES. We also assessed through simulations how aberrant expression and mono-allelic expression tests depend on RNA-seq coverage. RESULTS: We detected on average 12,500 genes per sample including around 60% of all disease genes-a coverage substantially higher than with whole blood, supporting the use of skin biopsies. We prioritized genes demonstrating aberrant expression, aberrant splicing, or mono-allelic expression. The pipeline required less than 1 week from sample preparation to result reporting and provided a median of eight disease-associated genes per patient for inspection. A genetic diagnosis was established for 16% of the 205 WES-inconclusive cases. Detection of aberrant expression was a major contributor to diagnosis including instances of 50% reduction, which, together with mono-allelic expression, allowed for the diagnosis of dominant disorders caused by haploinsufficiency. Moreover, calling aberrant splicing and variants from RNA-seq data enabled detecting and validating splice-disrupting variants, of which the majority fell outside WES-covered regions. CONCLUSION: Together, these results show that streamlined experimental and computational processes can accelerate the implementation of RNA-seq in routine diagnostics.

Whole genome and exome sequencing identify NDUFV2 mutations as a new cause of progressive cavitating leukoencephalopathy.

BACKGROUND: Progressive cavitating leukoencephalopathy (PCL) is thought to result from mutations in nuclear genes affecting mitochondrial function and energy metabolism. To date, mutations in two subunits of complex I, NDUFS1 and NDUFV1, have been reported to be related to PCL. METHODS: Patients underwent clinical examinations, brain MRI, skin biopsy and muscle biopsy. Whole-genome or whole-exome sequencing was performed on the index patients from two unrelated families with PCL. The effects of the mutations were examined through complementation of the NDUFV2 mutation by cDNA expression. RESULTS: The common clinical features of the patients in this study were recurring episodes of acute or subacute developmental regression that appeared in the first years of life, followed by gradual remissions and prolonged periods of stability. MRI showed leukoencephalopathy with multiple cavities. Three novel NDUFV2 missense mutations were identified in these families. Complex I deficiency was confirmed in affected individuals' fibroblasts and a muscle biopsy. Functional and structural analyses revealed that these mutations affect the structural stability and function of the NDUFV2 protein, indicating that defective NDUFV2 function is responsible for the phenotypes in these individuals. CONCLUSIONS: Here, we report the clinical presentations, neuroimaging and molecular and functional analyses of novel mutations in NDUFV2 in two sibling pairs of two Chinese families presenting with PCL. We hereby expand the knowledge on the clinical phenotypes associated with mutations in NDUFV2 and the genotypes causative for PCL.

Biallelic COA7-Variants Leading to Developmental Regression With Progressive Spasticity and Brain Atrophy in a Chinese Patient.

OBJECTIVE: The cytochrome c oxidase assembly factor 7 (COA7) gene encodes a protein localized to mitochondria that is involved in the assembly of mitochondrial respiratory chain complex IV. Here, we report the clinical, genetic and biochemical analysis of a female patient with suspected mitochondrial disorder and novel variants in COA7, that presented with a considerably different phenotype and age of onset than the five COA7 patients reported to date. METHODS: We performed trio-exome sequencing in the affected patient and both parents. To verify the pathogenicity of the detected variants in COA7, mitochondrial enzyme activities and oxygen consumption rate were investigated in fibroblasts of the patient and her parents. RESULTS: A Chinese girl was referred at 9 months of age with a history of developmental delay and regression since 3 months of age. In the following months, she lost previously acquired skills and developed progressive spasticity of the lower extremities. Trio-exome sequencing revealed compound heterzygous variants in COA7 (c.511G > A/p.Ala171Thr and c.566A > G/p.Asn189Ser). Functional validation experiments revealed isolated complex IV deficiency and a significantly reduced mitochondrial respiration rate in patient-derived fibroblasts. INTERPRETATION: Hitherto, characteristic features of COA7 patients were described as slowly progressing neuropathy and spinocerebellar ataxia, starting at the toddler age and progressing into adulthood. In contrast, our patient was reported to show developmental delay from 3 months of age, which was found to be due to a rapidly progressive encephalopathy and brain atrophy seen at 9 months of age. Unexpectedly, the genetic investigation revealed a COA7-associated mitochondrial disease, which was confirmed functionally. Thus, this report broadens the genetic and clinical spectrum of this heterogeneous mitochondriopathy and highlights the value of the presented unbiased approach.

Enzymatic photoelectrochemical bioassay based on hierarchical CdS/NiO heterojunction for glucose determination.

The design and development of a 3D hierarchical CdS/NiO heterojunction and its application in a self-powered cathodic photoelectrochemical (PEC) bioanalysis is introduced. Specifically, NiO nanoflakes (NFs) were in situ formed on carbon fibers via a facile liquid-phase deposition method followed by an annealing step and subsequent integration with CdS quantum dots (QDs). The glucose oxidase (GOx) was then coated on the photocathode to allow the determination of glucose. Under 5 W 410 nm LED light and at a working voltage of 0.0 V (vs. Ag/AgCl), this method can assay glucose concentrations down to 1.77×10-9 M. The linear range was 5×10-7 M to 1×10-3 M, and the relative standard deviation (RSD) was below 5%. The photocathodic biosensor achieved target detection with high sensitivity and selectivity. This work is expected to stimulate more passion in the development of innovative hierarchical heterostructures for advanced self-powered photocathodic bioanalysis. Design of 3D hierarchical CdS/NiO heterojunction and its application in a self-powered cathodic photoelectrochemical (PEC) bioanalysis.

Muscular pathological features in patients with myasthenia gravis.

OBJECTIVE: To analyze muscle histopathology of myasthenia gravis (MG) patients and further explore the underlying mechanism comparing with previous literature. MATERIALS AND METHODS: We analyzed the clinicopathological features of 8 MG patients who had muscle biopsy examinations. RESULTS: Eight patients with a diagnosis of MG were retrospectively recruited from the Chinese PLA General Hospital. One patient had positive anti-MuSK antibodies, 5 patients had positive anti-AChR antibodies (1 of whom had additional positive anti-Titin antibodies), and 2 patients were seronegative. Seronegative-MG presented normal muscle histology, occasionally with lipid deposition. Small angular atrophy (mainly in type II fibers) and necrosis in H & E stain were found in AChR-MG, furthermore, patterns of polymyositis (PM) could be found in AChR-MG with anti-Titin antibodies. Mitochondrial abnormalities were found only in MuSK-MG. CONCLUSION: Muscle histological abnormalities mimicking myopathy may be found in MG patients. Patients with different antibodies present with different muscle histopathology. PM pattern pathology is a special pattern of muscle histology in MG that should not be misdiagnosed. Our study has extended the muscle pathological features of MG in addition to deepening the understanding of MG.

Case Report: Diagnosis of Human Alveolar Echinococcosis via Next-Generation Sequencing Analysis.

Introduction: Alveolar echinococcosis (AE) is a rare parasitic disease caused by the infection of Echinococcus multilocularis. AE may mimic malignancy both in clinical presentation and radiological imaging, which is often misdiagnosed as metastatic tumor. Recently, next-generation sequencing (NGS) technologies are increasingly being used to address a diverse range of biological questions. Here, we describe a rare case of alveolar echinococcosis diagnosed by pan-pathogen screening, using next-generation sequencing. To the best of our knowledge, this is the first reported case of AE which was definitely diagnosed relying NGS of cerebrospinal fluid (CSF). Case Presentation: A 33-year-old man presented with repeat seizure and progressive headache for six months. Head magnetic resonance imaging (MRI) showed multiple masses with edema. Lung and abdominal computer tomography (CT) revealed multiple masses in bilateral lung, liver and the right adrenal gland. Bacterial, tuberculosis and fungal infection were excluded by CSF examination. Repeated target biopsy on the masses in the lung and liver showed as fibrous connective tissue without positive findings. NGS of CSF was performed and detected nucleic acid sequences of E. multilocularis. Consequently, the patient has accepted 1-year albendazole therapy. His case was followed up through imaging procedures. Conclusion: The next-generation sequencing of CSF is a reliable and sensitive diagnostic method for the detection of pathogenic microorganisms, and may allow the accurate diagnosis of alveolar echinococcosis. In view of this case, we recommend NGS as a potential tool for diagnosis of cerebral AE, especially if repeated biopsies are negative.

A novel homozygous mutation in ATP13A2 gene causing pure hereditary spastic paraplegia.

SPG78 is a subtype of hereditary spastic paraplegia(HSP) caused by ATP13A2 gene mutations. SPG78 was reported as complicated HSP in several cases, but was never associated with pure HSP. Here we report the first Chinese patient carrying a novel homozygous nonsense mutation in ATP13A2 presenting with pure HSP.