Anti-CRMP2 antibody induces anxiety-like behavior and increases pyramidal neuron excitability in mice.

BACKGROUND: We have previously identified auto-antibody (Ab) to collapsin response mediator protein 2 (CRMP2) in patients with encephalitis. The present study aims to evaluate the pathogenic effects of anti-CRMP2 Ab. METHODS: Recombinant CRMP2 protein was injected subcutaneously into mice to establish an active immune mouse model with anti-CRMP2 Ab. Behavioral assessments, histopathological staining, and electrophysiological testing were performed to identify any pathogenic changes. RESULTS: The mice exhibited signs of impaired motor coordination four weeks post-immunization of CRMP2 protein. Moreover, CRMP2 immunized mice for eight weeks showed anxiety-like behaviors indicating by tests of open field and the elevated plus maze. After incubating the CA1 region of hippocampal brain section with the sera from CRMP2 immunized mice, the whole-cell path-clamp recordings showed increased excitability of pyramidal neurons. However, no obvious inflammation and infiltration of immune cells were observed by histopathological analysis. Western blot showed that the phosphorylation levels of CRMP2-Thr514 and -Ser522 were not affected. CONCLUSION: In an active immunization model with CRMP2 protein, impaired coordination and anxiety-like behaviors were observed. Also, anti-CRMP2 Abs containing sera heightened the excitability of hippocampal pyramidal neurons in vitro, which imply the pathogenic effects of anti-CRMP2 Ab.

Neurosyphilis complicated by anti-γ-aminobutyric acid-B receptor encephalitis: A case report.

BACKGROUND: Syphilis is an infectious disease caused by Treponema pallidum that can invade the central nervous system, causing encephalitis. Few cases of anti-N-methyl-D-aspartate receptor autoimmune encephalitis (AE) secondary to neurosyphilis have been reported. We report a neurosyphilis patient with anti-γ-aminobutyric acid-B receptor (GABABR) AE. CASE SUMMARY: A young man in his 30s who presented with acute epileptic status was admitted to a local hospital. He was diagnosed with neurosyphilis, according to serum and cerebrospinal fluid (CSF) tests for syphilis. After 14 d of antiepileptic treatment and anti-Treponema pallidum therapy with penicillin, epilepsy was controlled but serious cognitive impairment, behavioral, and serious psychiatric symptoms were observed. He was then transferred to our hospital. The Mini-Mental State Examination (MMSE) crude test results showed only 2 points. Cranial magnetic resonance imaging revealed significant cerebral atrophy and multiple fluid-attenuated inversion recovery high signals in the white matter surrounding both lateral ventricles, left amygdala and bilateral thalami. Anti-GABABR antibodies were discovered in CSF (1:3.2) and serum (1:100). The patient was diagnosed with neurosyphilis complicated by anti-GABABR AE, and received methylprednisolone and penicillin. Following treatment, his mental symptoms were alleviated. Cognitive impairment was significantly improved, with a MMSE of 8 points. Serum anti-GABABR antibody titer decreased to 1:32. The patient received methylprednisolone and penicillin after discharge. Three months later, the patient's condition was stable, but the serum anti-GABABR antibody titer was 1:100. CONCLUSION: This patient with neurosyphilis combined with anti-GABABR encephalitis benefited from immunotherapy.

AKAP3-mediated type I PKA signaling is required for mouse sperm hyperactivation and fertility†.

The protein kinase A (PKA) signaling pathway, which mediates protein phosphorylation, is important for sperm motility and male fertility. This process relies on A-kinase anchoring proteins that organize PKA and its signalosomes within specific subcellular compartments. Previously, it was found that the absence of A-kinase anchoring protein 3 (AKAP3) leads to multiple morphological abnormalities in mouse sperm. But how AKAP3 regulates sperm motility is yet to be elucidated. AKAP3 has two amphipathic domains, here named dual and RI, in its N-terminus. These domains are responsible for binding regulatory subunits I alpha (RIα) and II alpha (RIIα) of PKA and for RIα only, respectively. Here, we generated mutant mice lacking the dual and RI domains of AKAP3. It was found that the deletion of these domains caused male mouse infertile, accompanied by mild defects in the fibrous sheath of sperm tails. Additionally, the levels of serine/threonine phosphorylation of PKA substrates and tyrosine phosphorylation decreased in the mutant sperm, which exhibited a defect in hyperactivation under capacitation conditions. The protein levels of PKA subunits remained unchanged. But, interestingly, the regulatory subunit RIα was mis-localized from principal piece to midpiece of sperm tail, whereas this was not observed for RIIα. Further protein-protein interaction assays revealed a preference for AKAP3 to bind RIα over RIIα. Collectively, our findings suggest that AKAP3 is important for sperm hyperactivity by regulating type-I PKA signaling pathway mediated protein phosphorylation via its dual and RI domains.

Discovery and Development of 4-Hydroxyphenylpyruvate Dioxygenase as a Novel Crop Fungicide Target.

Plant pathogenic fungi pose a significant threat to crop yields and quality, and the emergence of fungicide resistance has further exacerbated the problem in agriculture. Therefore, there is an urgent need for efficient and environmentally friendly fungicides. In this study, we investigated the antifungal activity of (+)-Usnic acid and its inhibitory effect on crop pathogenic fungal 4-hydroxyphenylpyruvate dioxygenases (HPPDs) and determined the structure of Zymoseptoria tritici HPPD (ZtHPPD)-(+)-Usnic acid complex. Thus, the antifungal target of (+)-Usnic acid and its inhibitory basis toward HPPD were uncovered. Additionally, we discovered a potential lead fungicide possessing a novel scaffold that displayed remarkable antifungal activities. Furthermore, our molecular docking analysis revealed the unique binding mode of this compound with ZtHPPD, explaining its high inhibitory effect. We concluded that HPPD represents a promising target for the control of phytopathogenic fungi, and the new compound serves as a novel starting point for the development of fungicides and dual-purpose pesticides.

Backbone Cyclization of Flavin Mononucleotide-Based Fluorescent Protein Increases Fluorescence and Stability.

Flavin mononucleotide-binding proteins or domains emit cyan-green fluorescence under aerobic and anaerobic conditions, but relatively low fluorescence and less thermostability limit their application as reporters. In this work, we incorporated the codon-optimized fluorescent protein from Chlamydomonas reinhardtii with two different linkers independently into the redox-responsive split intein construct, overexpressed the precursors in hyperoxic Escherichia coli SHuffle T7 strain, and cyclized the target proteins in vitro in the presence of the reducing agent. Compared with the purified linear protein, the cyclic protein with the short linker displayed enhanced fluorescence. In contrast, cyclized protein with incorporation of the long linker including the myc-tag and human rhinovirus 3C protease cleavable sequence emitted slightly increased fluorescence compared with the protein linearized with the protease cleavage. The cyclic protein with the short linker also exhibited increased thermal stability and exopeptidase resistance. Moreover, induction of the target proteins in an oxygen-deficient culture rendered fluorescent E. coli BL21 (DE3) cells brighter than those overexpressing the linear construct. Thus, the cyclic reporter can hopefully be used in certain thermophilic anaerobes.

Tuberculostearic acid incorporated predictive model contributes to the clinical diagnosis of tuberculous meningitis.

The conventional confirmation tests of tuberculous meningitis (TBM) are usually low in sensitivity, leading to high TBM mortality. Hence, sensitive methods for indicating the presence of bacilli are required. Tuberculostearic acid (TBSA), a constituent from Mycobacterium tuberculosis had been evaluated as a promising marker, but fails to demonstrate consistent results for definite TBM. This study retrospectively reviewed medical records of 113 TBM suspects, constructing a TBSA-combined scoring system based on multiple factors, which show sensitivity and specificity of 0.8148 and 0.8814, respectively, and the area under the receiver operating characteristic curve of 0.9010. Multivariate analyses revealed four co-predictive factors strongly associated with TBSA: extra-neural tuberculosis, basal meningeal enhancement, CSF glucose/Serum glucose <0.595, and coinfection in CNS (Total). The subsequent machine learning-based validation showed correspondent importance to factors in the TBSA model. This study demonstrates a simple scoring system to facilitate TBM prediction, yield reliable diagnoses and allow timely treatment initiation.

Secondary immunoreaction in patients with neurosyphilis and its relevance to clinical outcomes.

Background and purpose: Several reported cases of autoimmune conditions such as anti-NMDAR encephalitis and neuromyelitis optica (AQP4) have been considered to be potentially secondary to Treponema pallidum infection. Since the role of immune impairment in neurosyphilis is unclear, in this retrospective study, we examined the correlation of the immune impairment in patients with neurosyphilis with their clinical characteristics and outcomes. Methods: Clinical information was collected from patients with neurosyphilis in our center from January 2019 to December 2021. Cerebrospinal fluid (CSF) samples were subjected to indirect immunofluorescence tissue-based assay (IIF-TBA) on mouse brain sections and cell-based assay (CBA). The clinical characteristics and treatment outcomes of TBA-positive and-negative patients were compared. Results: A total number of 81 patients diagnosed with neurosyphilis were included. The results of the CBA tests showed that three cases had anti-NMDAR, AQP4, or GAD65 antibodies, respectively. By TBA test, 38 patients (38/81, 46.9%) had positive immunostains, including staining of neuronal cells in 21 cases (21/38, 55.3%), glial cells in 11 cases (11/38, 28.9%), and neuronal and glial cells in six cases (6/38, 15.8%). We then compared the clinical characteristics and treatment outcomes between the TBA-positive and-negative patients and found that TBA-positive staining was significantly correlated with syphilis antibody titers (p = 0.027 for serum and p = 0.006 for CSF) and head MRI abnormalities (p < 0.001 for parenchymal abnormalities and p = 0.013 for white matter lesions). The cognitive prognosis of TBA-positive neurosyphilis patients was significantly worse than that of TBA-negative patients (p < 0.001). Conclusion: The correlation between the TBA results and clinical data of our neurosyphilis patients imply the presence of secondary immune damage, which affected their prognosis. Therefore, TBA can be used as an additional biomarker for neurosyphilis patient prognosis.

TRIM32 biallelic defects cause limb-girdle muscular dystrophy R8: identification of two novel mutations and investigation of genotype-phenotype correlation.

BACKGROUND: Limb-girdle muscular dystrophy R8 (LGMD R8) is a rare autosomal recessive muscle disease caused by TRIM32 gene biallelic defects. The genotype-phenotype correlation of this disease has been reported poorly. Here, we report a Chinese family with two female LGMD R8 patients. METHODS: We performed whole-genome sequencing (WGS) and Sanger sequencing on the proband. Meanwhile, the function of mutant TRIM32 protein was analyzed by bioinformatics and experimental analysis. In addition, a summary of the reported TRIM32 deletions and point mutations and an investigation of genotype-phenotype correlation were performed through a combined analysis of the two patients and other cases reported in previous literature. RESULTS: The two patients displayed typical symptoms of LGMD R8, which worsened during pregnancy. Genetic analysis by whole-genome sequencing (WGS) and Sanger sequencing showed that the patients were compound heterozygotes of a novel deletion (chr9.hg19:g.119431290_119474250del) and a novel missense mutation (TRIM32:c.1700A > G, p.H567R). The deletion encompassed 43 kb and resulted in the removal of the entire TRIM32 gene. The missense mutation altered the structure and further affected function by interfering with the self-association of the TRIM32 protein. Females with LGMD R8 showed less severe symptoms than males, and patients carrying two mutations in NHL repeats of the TRIM32 protein had earlier disease onset and more severe symptoms than other patients. CONCLUSIONS: This research extended the spectrum of TRIM32 mutations and firstly provided useful data on the genotype-phenotype correlation, which is valuable for the accurate diagnosis and genetic counseling of LGMD R8.

Use of the redox-dependent intein system for enhancing production of the cyclic green fluorescent protein.

To expand the reported redox-dependent intein system application, in this work, we used the split intein variant with highly trans-splicing efficiency and minimal extein dependence to cyclize the green fluorescent protein variant reporter in vitro. The CPG residues were introduced adjacent to the intein's catalytic cysteine for reversible formation of a disulfide bond to retard the trans-splicing reaction under the oxidative environment. The cyclized reporter protein in Escherichia coli cells was easily prepared by organic extraction and identified by the exopeptidase digestion. The amounts of extracted cyclized protein reporter in BL21 (DE3) cells were higher than those in hyperoxic SHuffle T7 coexpression system for facilitating the disulfide bond formation. The double His6-tagged precursor was purified for in vitro cyclization of the protein for 3 h. Compared with the purified linear counterpart, the cyclic reporter showed about twofold increase in fluorescence intensity, exhibited thermal and hydrolytic stability, and displayed better folding efficiency in BL21 (DE3) cells at the elevated temperature. Taken together, the developed redox-dependent intein system will be used for producing other cyclic disulfide-free proteins. The cyclic reporter is a potential candidate applied in certain thermophilic aerobes.

Reduced mitochondrial size in hippocampus and psychiatric behavioral changes in the mutant mice with homologous mutation of Timm8a1-I23fs49X.

Background: Deafness-dystonia-optic neuronopathy (DDON) syndrome, a condition that predominantly affects males, is caused by mutations in translocase of mitochondrial inner membrane 8A (TIMM8A)/deafness dystonia protein 1 (DDP1) gene and characterized by progressive deafness coupled with other neurological abnormalities. In a previous study, we demonstrated the phenotype of male mice carrying the hemizygous mutation of Timm8a1-I23fs49X. In a follow-up to that study, this study aimed to observe the behavioral changes in the female mutant (MUT) mice with homologous mutation of Timm8a1 and to elucidate the underlying mechanism for the behavioral changes. Materials and methods: Histological analysis, transmission electron microscopy (EM), Western blotting, hearing measurement by auditory brainstem response (ABR), and behavioral observation were compared between the MUT mice and wild-type (WT) littermates. Results: The weight of the female MUT mice was less than that of the WT mice. Among MUT mice, both male and female mice showed hearing impairment, anxiety-like behavior by the elevated plus maze test, and cognitive deficit by the Morris water maze test. Furthermore, the female MUT mice exhibited coordination problems in the balance beam test. Although the general neuronal loss was not found in the hippocampus of the MUT genotype, EM assessment indicated that the mitochondrial size showing as aspect ratio and form factor in the hippocampus of the MUT strain was significantly reduced compared to that in the WT genotype. More importantly, this phenomenon was correlated with the upregulation of translation of mitochondrial fission process protein 1(Mtfp1)/mitochondrial 18 kDa protein (Mtp18), a key fission factor that is a positive regulator of mitochondrial fission and mitochondrial size. Interestingly, significant reductions in the size of the uterus and ovaries were noted in the female MUT mice, which contributed to significantly lower fertility in the MUT mice. Conclusion: Together, a homologous mutation in the Timm8a1 gene caused the hearing impairment and psychiatric behavioral changes in the MUT mice; the latter phenotype might be related to a reduction in mitochondrial size regulated by MTP18.

Chicken jejunal microbiota improves growth performance by mitigating intestinal inflammation.

BACKGROUND: Intestinal inflammation is prevalent in chicken, which results in decreased growth performance and considerable economic losses. Accumulated findings established the close relationship between gut microbiota and chicken growth performance. However, whether gut microbiota impacts chicken growth performance by lessening intestinal inflammation remains elusive. RESULTS: Seven-weeks-old male and female chickens with the highest or lowest body weights were significantly different in breast and leg muscle indices and average cross-sectional area of muscle cells. 16S rRNA gene sequencing indicated Gram-positive bacteria, such as Lactobacilli, were the predominant species in high body weight chickens. Conversely, Gram-negative bacteria, such as Comamonas, Acinetobacter, Brucella, Escherichia-Shigella, Thermus, Undibacterium, and Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium were significantly abundant in low body weight chickens. Serum lipopolysaccharide (LPS) level was significantly higher in low body weight chickens (101.58 ± 5.78 ng/mL) compared with high body weight chickens (85.12 ± 4.79 ng/mL). The expression of TLR4, NF-κB, MyD88, and related inflammatory cytokines in the jejunum was significantly upregulated in low body weight chickens, which led to the damage of gut barrier integrity. Furthermore, transferring fecal microbiota from adult chickens with high body weight into 1-day-old chicks reshaped the jejunal microbiota, mitigated inflammatory response, and improved chicken growth performance. CONCLUSIONS: Our findings suggested that jejunal microbiota could affect chicken growth performance by mitigating intestinal inflammation. Video Abstract.

Identification of Anti-Collapsin Response Mediator Protein 2 Antibodies in Patients With Encephalitis or Encephalomyelitis.

Background and Purpose: An increasing number of autoimmune encephalitis (AE)-associated autoantibodies have been successfully characterized. However, many cases of AE remain unexplained on account of unknown antibodies. The aim of the present study was to identify a novel antibody against collapsin response mediator protein 2 (CRMP2) in suspected AE patients. Methods: A patient's serum and cerebrospinal fluid samples tested negative for known AE antibodies; however, strong immunolabel signals were observed in the neuronal cytoplasm of the cortex, hippocampus, and Purkinje cells on rat brain sections. Immunoprecipitation from the rat brain protein lysate, followed by mass spectrometry analysis, was used to identify the targeting antigen. Western blotting and cell-based assay with antigen-overexpressing HEK293T cells were used for antibody specificity, epitope, IgG subtype determination, and retrospective study. Results: An antibody against CRMP2, a synaptic protein involved in axon guidance, was identified. The immunostains of the patient's samples on rat brain sections were eliminated by pre-absorption with HEK293T cells overexpressing CRMP2. The samples specifically immunoreacted with CRMP2, but not with CRMP1, CRMP3, CRMP4, and CRMP5. The C-terminus of CRMP2 with 536 amino acids contained the epitope for antibody binding. The subtype analysis showed that the anti-CRMP2 antibody was IgG4. Furthermore, a screening of 46 patients with neurological disoders and neuro-cytoplasm immunostainings on rat brain sections resulted in the identification of anti-CRMP2 antibodies in a case of encephalomyelitis. The two patients responded well to immunotherapies. Conclusions: This study discovered that a novel anti-CRMP2 antibody was associated with suspected AE and thus should be included in the testing list for AE.

Two CIDP Variants Patients With Anti-Caspr1 Antibodies in South China.

Background and Objectives: Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is considered an immune-mediated heterogeneous disease that involves both cellular and humoral immunity. The advent of the new concept of node-paranodopathy in recent years has boosted the identification of more antibody-positive CIDP variants patients. Cases of Caspr1 autoantibodies are the least common. Here, we reported two patients with Caspr1 autoantibodies and summarized their clinical features and treatment responses. Methods: Do statistical analyses on the clinical manifestations and laboratory examinations obtained from two patients identified in this study, and eight patients with anti-Caspr1 antibodies reported in previous research. And based on the developed scoring standard, draw the radar charts and line graphs. Results: Similar to other studies, the two patients we mentioned had a subacute and severe onset, distal phenotype, sensory ataxia, and severe pain. Differently, they had severe pain accompanying cold sense and coarse tremor in both hands, which may be a typical symptom for the anti-Caspr1 positive patient in south China. And we drew the line and radar graph for two China patients based on five aspects, muscle strength, sensory nerve, cranial nerve, laboratory tests, and NCS examinations. The two visual data charts offered new complementary means for the diagnostic assessment of CIDP variants. Conclusion: Pain with cold sense, coarse tremor in hands, and CSF protein levels greater than 3g/L may be the source of the distinct symptoms observed in patients with anti-Caspr1 autoantibodies in south China.

Caecal microbiota could effectively increase chicken growth performance by regulating fat metabolism.

It has been established that gut microbiota influences chicken growth performance and fat metabolism. However, whether gut microbiota affects chicken growth performance by regulating fat metabolism remains unclear. Therefore, seven-week-old chickens with high or low body weight were used in the present study. There were significant differences in body weight, breast and leg muscle indices, and cross-sectional area of muscle cells, suggesting different growth performance. The relative abundance of gut microbiota in the caecal contents at the genus level was compared by 16S rRNA gene sequencing. The results of LEfSe indicated that high body weight chickens contained Microbacterium and Sphingomonas more abundantly (P < 0.05). In contrast, low body weight chickens contained Slackia more abundantly (P < 0.05). The results of H & E, qPCR, IHC, WB and blood analysis suggested significantly different fat metabolism level in serum, liver, abdominal adipose, breast and leg muscles between high and low body weight chickens. Spearman correlation analysis revealed that fat metabolism positively correlated with the relative abundance of Microbacterium and Sphingomonas while negatively correlated with the abundance of Slackia. Furthermore, faecal microbiota transplantation was performed, which verified that transferring faecal microbiota from adult chickens with high body weight into one-day-old chickens improved growth performance and fat metabolism in liver by remodelling the gut microbiota. Overall, these results suggested that gut microbiota could affect chicken growth performance by regulating fat metabolism.

Acrolein, an endogenous aldehyde induces Alzheimer's disease-like pathologies in mice: A new sporadic AD animal model.

Alzheimer's disease (AD) is a common neurodegenerative disease that mainly affects elderly people. However, the translational research of AD is frustrating, suggesting that the development of new AD animal models is crucial. By gavage administration of acrolein, we constructed a simple sporadic AD animal model which showed classic pathologies of AD in 1 month. The AD-like phenotypes and pathological changes were as followed. 1) olfactory dysfunctions, cognitive impairments and psychological symptoms in C57BL/6 mice; 2) increased levels of Aß1-42 and Tau phosphorylation (S396/T231) in cortex and hippocampus; 3) astrocytes and microglia proliferation; 4) reduced levels of postsynaptic density 95(PSD95) and Synapsin1, as well as the density of dendritic spines in the CA1 and DG neurons of the hippocampus; 5) high-frequency stimulation failed to induce the long-term potentiation (LTP) in the hippocampus after exposure to acrolein for 4 weeks; 6) decreased blood oxygen level-dependent (BOLD) signal in the olfactory bulb and induced high T2 signals in the hippocampus, which matched to the clinical observation in the brain of AD patients, and 7) activated RhoA/ROCK2/ p-cofilin-associated pathway in hippocampus of acrolein-treated mice, which may be the causes of synaptic damage and neuroinflammation in acrolein mice model. Taken together, the acrolein-induced sporadic AD mouse model closely reflects the pathological features of AD, which will be useful for the research on the mechanism of AD onset and the development of anti-AD drugs.

Charcot-Marie-Tooth Disease With Episodic Rhabdomyolysis Due to Two Novel Mutations in the ß Subunit of Mitochondrial Trifunctional Protein and Effective Response to Modified Diet Therapy.

A 29-year-old female experienced chronic progressive peripheral neuropathy since childhood and was diagnosed with Charcot-Marie-Tooth disease (CMT) at age 15. She developed recurrent, fever-induced rhabdomyolysis (RM) at age 24. EMG studies showed decreased amplitude of compound muscle action potential, declined motor conductive velocity, and absence of sensor nerve action potential. Acylcarnitine analysis revealed elevated C16-OH, C18-OH, and C18:1-OH. Muscle biopsy showed scattered foci of necrotic myofibers invaded by macrophages, occasional regenerating fibers, and remarkable muscle fiber type grouping. Whole-exome sequencing identified two novel heterozygous mutations: c.490G>A (p.G164S) and c.686G>A (p.R229Q) in HADHB gene encoding the ß-subunit of mitochondrial trifunctional protein (MTP). Reduction of long-chain fatty acid via dietary restrictions alleviated symptoms effectively. Our study indicates that the defect of the MTP ß-subunit accounts for both CMT and RM in the same patient and expands the clinical spectrum of disorders caused by the HADHB mutations. Our systematic review of all MTPD patients with dietary treatment indicates that the effect of dietary treatment is related to the age of onset and the severity of symptoms.

Construction, Investigation and Application of TEV Protease Variants with Improved Oxidative Stability.

Tobacco etch virus protease (TEVp) is a useful tool for removing fusion tags, but wild-type TEVp is less stable under oxidized redox state. In this work, we introduced and combined C19S, C110S and C130S into TEVp variants containing T17S, L56V, N68D, I77V and S135G to improve protein solubility, and S219V to inhibit self-proteolysis. The solubility and cleavage activity of the constructed variants in Escherichia coli strains including BL21(DE3), BL21(DE3)pLys, Rossetta(DE3) and Origami(DE3) under the same induction conditions were analyzed and compared. The desirable soluble amounts, activity, and oxidative stability were identified to be reluctantly favored in the TEVp. Unlike C19S, C110S and C130S hardly impacted on decreasing protein solubility in the BL21(DE3), but they contributed to improved tolerance to the oxidative redox state in vivo and in vitro. After two fusion proteins were cleaved by purified TEVp protein containing double mutations under the oxidized redox state, the refolded disulfide-rich bovine enterokinase catalytic domain or maize peroxidase with enhanced yields were released from the regenerated amorphous cellulose via affinity absorption of the cellulose-binding module as the affinity tag.

Molecular epidemiology of coxsackievirus A16 circulating in children in Beijing, China from 2010 to 2019.

BACKGROUND: Coxsackievirus A16 (CVA16) is one of the major etiological agents of hand, foot and mouth disease (HFMD). This study aimed to investigate the molecular epidemiology and evolutionary characteristics of CVA16. METHODS: Throat swabs were collected from children with HFMD and suspected HFMD during 2010-2019. Enteroviruses (EVs) were detected and typed by real-time reverse transcription-polymerase chain reaction (RT-PCR) and RT-PCR. The genotype, evolutionary rate, the most recent common ancestor, population dynamics and selection pressure of CVA16 were analyzed based on viral protein gene (VP1) by bioinformatics software. RESULTS: A total of 4709 throat swabs were screened. EVs were detected in 3180 samples and 814 were CVA16 positive. More than 81% of CVA16-positive children were under 5 years old. The prevalence of CVA16 showed obvious periodic fluctuations with a high level during 2010-2012 followed by an apparent decline during 2013-2017. However, the activities of CVA16 increased gradually during 2018-2019. All the Beijing CVA16 strains belonged to sub-genotype B1, and B1b was the dominant strain. One B1c strain was detected in Beijing for the first time in 2016. The estimated mean evolutionary rate of VP1 gene was 4.49 × 10-3 substitution/site/year. Methionine gradually fixed at site-23 of VP1 since 2012. Two sites were detected under episodic positive selection, one of which (site-223) located in neutralizing linear epitope PEP71. CONCLUSIONS: The dominant strains of CVA16 belonged to clade B1b and evolved in a fast evolutionary rate during 2010-2019 in Beijing. To provide more favorable data for HFMD prevention and control, it is necessary to keep attention on molecular epidemiological and evolutionary characteristics of CVA16.

Combination of the mutations for improving activity of TEV protease in inclusion bodies.

Tobacco etch virus protease (TEVp) is an enzymatic reagent to remove fusion tag, but additional purification steps are required for removing the TEVp after cleavage reaction is finished. Use of carrier-free and dependent TEVp immobilizates can eliminate protease contamination. In this work, we identified that, among the four constructed missense variants, the insoluble variant with the highest activity was correspondent with the soluble one tested formerly. The activities of the insoluble 15 codon variants were assayed and the variant with highest activity was selected. The K45F and/or E106G mutations have been reported on slightly improving protein stability of the wild-type TEVp, but only E106G mutation enhanced soluble production and activity of the selected TEVp variant, and it increased soluble amounts of two codon variants with the impaired folding. The decreased activity and use efficiency of the optimized TEVp variant in inclusion bodies was balanced by the determined high level production, lower leaking amounts of the protein, the enhanced resistance to the limited proteolysis mediated by protease K and trypsin, and the increased inhibition of auto-cleavage, as comparison to those of the immobilized soluble one. Thus, the TEVp construct is a potential alternate for simplifying protein purification protocols after tag-removal.