Neuroprotective effects of an engineered commensal bacterium in the 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine Parkinson disease mouse model via producing glucagon-like peptide-1.

While glucagon-like peptide-1 (GLP-1) was reported to have a positive impact on Parkinson disease, it is extremely short half-life greatly hindered its clinical use. In this study, the mouse strain MG1363-pMG36e-GLP-1 was engineered to continuously express GLP-1 to treat Parkinson disease in a 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-treated Parkinson disease model. In our study, oral supplementation with MG1363-pMG36e-GLP-1 significantly (p < 0.05) reduced MPTP-induced locomotor impairments, increased tyrosine hydroxylase-positive neurons, suppressed microglia and astrocyte activation, and down-regulated expression of several inflammation-related molecules. In addition, MG1363-pMG36e-GLP-1 significantly (p < 0.01) reduced intestinal pathogen Enterobacteriaceae and markedly enhanced the number of probiotic Lactobacillus and Akkermansia. These data suggest that MG1363-pMG36e-GLP-1 could be a novel therapeutic means for Parkinson disease.

Triptolide modulates tumour-colonisation and anti-tumour effect of attenuated Salmonella encoding DNase I.

The strong human immunity and the associated toxicities of attenuated Salmonella severely limit the clinical use of Salmonella in tumour suppression. In the present study, we constructed an engineered VNP20009-DNase I strain and evaluated the synergistic effects of triptolide (TPL) and VNP20009-DNase I against melanoma in mice. Our results indicated that TPL could significantly inhibit the cell growth and cell migration and significantly enhanced the apoptosis rate of B16F10 cells in vitro. The in vivo results indicated that TPL markedly improved tumour colonisation of VNP20009-DNase I and led to a larger necrotic area in the melanoma. Moreover, the combination therapy significantly suppressed tumour volume and prolonged the life span of mice (P < 0.05) by upregulating the expression of Bcl-2/Bax and Caspase-3 and by downregulating the TLR4/NF-κB signalling, the expression of p-AKT/AKT and the production of proinflammatory factors. Therefore, the sound synergistic anti-tumour effects of TPL and VNP20009-DNase I indicate that the unconventional application of TPL and biological agents, approved by the China Food and Drug Administration (CFDA), can result in improved anti-cancer therapeutic outcomes.

Evaluation of truncated G protein delivered by live attenuated Salmonella as a vaccine against respiratory syncytial virus.

Human respiratory syncytial virus (RSV) can cause severe acute lower respiratory tract disease leading to numerous hospitalizations and deaths in the infant and elderly populations worldwide, while no vaccine or effective drug is available for RSV infections. In the present study, truncated G protein was successfully expressed both in prokaryotic and eukaryotic system, and high levels of serum IgG in response to truncated G protein were observed both in GD-protein group (intramuscularly with purified GD protein) and GD-VNP20009 group (challenged via the oral route with 1 × 109 CFU of pLIVE-RSV-GD-VNP20009 strains) since 21th day, and GD-VNP20009 significantly reduced the productions of IL-1ß, IL-6, and TNF-α, histamine and pathological features caused by the RSV Long strain (P < .01). Our data indicated that Salmonella typhimurium can be used to deliver truncated G DNA vaccine and represents a promising effect to protect host against RSV.

Engineered commensal bacteria prevent systemic inflammation-induced memory impairment and amyloidogenesis via producing GLP-1.

The anti-obesity drug GLP-1 has been proven to have an impact on central nervous system, while its extremely short half-life greatly limited its use. In this study, our group constructed two engineering strains MG1363-pMG36e-GLP-1 and VNP20009-pLIVE-GLP-1 to continuously express GLP-1, and supplementation of these strains, especially MG1363-pMG36e-GLP-1, had significantly restored the spatial learning and memory impairment of mice caused by LPS (p < 0.05), suppressed glia activation and Aß accumulation, and downregulated inflammatory expressions of COX-2, TLR-4, TNF-a, and IL-1ß. In addition, MG1363-pMG36e-GLP-1 had significantly blocked the translocation of NF-κB signal and inhibited the phosphorylation of redox-sensitive cytoplasmic signalings of MAPKs and PI3K/AKT. These data suggest that MG1363-pMG36e-GLP-1 could be used as a safe and effective nonabsorbed oral treatment for neuroinflammation-related diseases such as Alzheimer's disease (AD).

Reclamation of Herb Residues Using Probiotics and Their Therapeutic Effect on Diarrhea.

Residues from herbal medicine processing in pharmaceutical plants create a large amount of waste (herb residues), which consists mainly of environmental pollution and medicinal waste. In order to resolve this problem, probiotics of Bacillus (B.) subtilis, Aspergillus (A.) oryzae, and Lactobacillus (L.) plantarum M3 are selected to reuse herb residue of Jianweixiaoshi tablets (JT), and an antibiotic-associated diarrhea (AAD) mouse model was established to evaluate the therapeutic effects of the herb residue fermentation supernatant. Our results indicated that the fermentation supernatant had scavenged 77.8% of 2,2-diphenyl-1-picrylhydrazyl (DPPH), 78% of O2•-, 36.7% of •OH, 39% of Fe2+ chelation, and 716 mg/L reducing power. The inhibition zones for Salmonella (S.) typhimurium, S. enteritidis, Shigella (Sh.) flexneri, Escherichia (E.) coli, Listeria (L.) monocytogenes, Sh. dysenteriae 301, and Staphylococcus (S.) aureus were 17, 14, 19, 18, 20, 19, and 20 mm, respectively. The in vivo results indicated that the fermentation supernatant resulted in a high diarrhea inhibition rate (56%, p < 0.05), greatly enhanced the disruption of bacterial diversity caused by antibiotics, and restored the dominant position of L. johnsonii in the treatment and recovery stages. Therefore, the combination of the herb residue and probiotics suggests a potential to explore conversion of these materials for the possible development of therapies for AAD.

Combination of thalidomide and Clostridium butyricum relieves chemotherapy-induced nausea and vomiting via gut microbiota and vagus nerve activity modulation.

Nausea and vomiting (CINV) are distressful and widespread side effects of chemotherapy, and additional efficient regimens to alleviate CINV are urgently needed. In the present study, colorectal cancer (CRC) mice model induced by Azoxymethane (AOM)/Dextran Sodium Sulfate (DSS) was employed to evaluate the cancer suppression and CINV amelioration effect of the combination of thalidomide (THD) and Clostridium butyricum. Our results suggested that the combination of THD and C. butyricum abundantly enhanced the anticancer effect of cisplatin via activating the caspase-3 apoptosis pathway, and also ameliorated CINV via inhibiting the neurotransmitter (e.g., 5-HT and tachykinin 1) and its receptor (e.g., 5-HT3R and NK-1R) in brain and colon. Additionally, the combination of THD and C. butyricum reversed the gut dysbacteriosis in CRC mice by increasing the abundance of Clostridium, Lactobacillus, Bifidobacterium, and Ruminococcus at the genus level, and also led to increased expression of occludin and Trek1 in the colon, while decreased expression of TLR4, MyD88, NF-κB, and HDAC1, as well as the mRNA level of IL-6, IL-1ß, and TNF-α. In all, these results suggest that the combination of THD and C. butyricum had good efficacy in enhancing cancer treatments and ameliorating CINV, which thus provides a more effective strategy for the treatment of CRC.

Effects of Yellow Light on Airborne Microbial Composition and on the Transcriptome of Typical Marker Strain in Ward.

Airborne diseases are transmitted by pathogens in the air. The complex microbial environment in wards is usually considered a major cause of nosocomial infection of various diseases which greatly influences the health of patients with chronic diseases, whereas the illuminant of wards impacts on the microbe especially the disease marker strain is seldom studied. In the present study, high-throughput sequencing was used to study the effect of yellow light on airborne microbial composition, and changes of transcriptome of marker strains Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa, which were isolated from wards, were further studied after the irradiation by yellow light. High-throughput sequencing results indicated that yellow light significantly decreased α-diversity. The relative abundance of Firmicutes at the phylum level, and Clostridium sensu stricto 1, Paraclostridium at the genus level were significantly reduced. RNA sequencing results declared that yellow light significantly downregulated the genes associated with flagella, heme transport system and carbohydrate, amino acid metabolism in E. coli, and the genes related to arginine biosynthesis and the biosynthesis of isoleucine, leucine, and valine in S. aureus. Meanwhile, yellow light significantly upregulated the genes relating to porphyrin metabolism in P. aeruginosa. In conclusion, our work reveals the impacts of yellow light on the microbe in wards, pointing out the application value of yellow light in the prevention of infectious diseases in clinical practice.

Fecal Microbiota Transplantation Could Improve Chronic Diarrhea in Cynomolgus Monkey by Alleviating Inflammation and Modulating Gut Microbiota.

Chronic diarrhea is associated with enteric dysbiosis and provokes the overuse of antibiotics. Fecal microbiota transplantation (FMT) is a promising therapy, but it shows discrepant clinical efficacy. Bacterial colonization in recipients has been studied, although little is known about the role of gut fungi and Archaea after FMT. In this study, we evaluated the efficacy of human-derived FMT on spontaneous chronic diarrhea cynomolgus monkeys and revealed the effector mechanisms. We demonstrated that FMT can mitigate the appearance of diarrheal symptoms and inhibit the increase in interleukin-6, interleukin-8, interleukin-1ß, and interferon-γ and the decrease in interleukin-10 in serum. We confirmed that FMT restored the disturbance of gut bacteria by reducing the relative abundances of potential pathogens, including Cloacibacillus porcorum, Desulfovibrio desulfuricans, Erysipelotrichaceae bacterium 5_2_54FAA, and Erysipelotrichaceae bacterium 21_3, and increasing the levels of Lactobacillus fermentum and Lactobacillus ruminis CAG_367 in diarrheal monkeys. The metabolic pathways of healthy and FMT monkeys' gut bacteria were enriched in amino acid metabolism, carbohydrate metabolism, and lipid metabolism, while the metabolic pathways of pre-FMT monkeys' gut bacteria were enriched in antibiotic production. Moreover, a higher Ascomycota/Basidiomycota ratio, higher Aspergillus levels, and lower Trichosporon asahii abundance were present in intestinal fungi after FMT. Although the abundance of the Archaea Methanosphaera stastmanae did not change significantly, it was inversely correlated with the anti-inflammatory factor IL-4 after FMT. These results support the further development and application of FMT for chronic diarrhea.

Combination of an engineered Lactococcus lactis expressing CXCL12 with light-emitting diode yellow light as a treatment for scalded skin in mice.

Impaired wound closure is an increasingly crucial clinical challenge. Recently, wound healing has shifted towards innovative treatments that exploit nanotechnology, biomaterials, biologics and phototherapy. Here, we constructed an engineered MG1363-pMG36e-mCXCL12 strain with pMG36e plasmid encoding stromal cell-derived factor 1α (named CXCL12) and evaluated the synergistic effects of light-emitting diode (LED) yellow light and MG1363-pMG36e-mCXCL12 on scald wounds in mice. The results indicated that the combined treatment with LED yellow light with mCXCL12 delivering strain accelerated wound closure, tissue remodelling, re-epithelialization and hair follicle regeneration and inhibited over-inflammation oppositely in the central and surrounding wounds by macroscopic, histopathologic and immunohistochemistry parameters. Furthermore, combination therapy increased the epidermal growth factor and Ki67-positive cells and upregulated beta-catenin (ß-catenin), cellular-myelocytomatosis (c-Myc), wingless-type MMTV integration site family member 1 (Wnt1), Jagged 1, neurogenic locus notch homolog protein 1 (Notch 1) and hairy and enhancer of split 1 (Hes 1) protein levels of the Wnt and Notch signalling pathways. It also facilitated collagen fibrogenesis and deposition and improved the activities of hydroxyproline, superoxide dismutase and glutathione peroxidase in scalded granulation tissue, in addition to reducing the inflammatory factors interleukin 1 beta (IL-1ß) and tumour necrosis factor alpha (TNF-α). The combined treatment effectively reduced skin pathogens Ralstonia and Acinetobacter to further reduce the risk of infection. Overall, combination of LED yellow light and MG1363-pMG36e-mCXCL12 represents a potential strategy for the treatment of cutaneous wounds.

GTW inhibits the Epithelial to Mesenchymal Transition of Epithelial Ovarian Cancer via ILK/AKT/GSK3ß/Slug Signalling Pathway.

Background: Epithelial ovarian cancer (EOC) accounts for the most lethal of all gynaecological cancers which is attributed to metastasis, invasiveness and drug resistance. A crucial link has been found between epithelial-mesenchymal transition (EMT) and cancer metastasis and chemo-resistance. Previous studies have confirmed that one of the main components of tripterygium glycosides (GTW)-triptolide (TPL) has anticancer effects. Methods: The purpose of this study is to determine whether GTW could inhibit EMT in A2780/DPP cells in vitro and in vivo, and explore the underlying mechanism. Results: In vitro results showed that GTW inhibited cell proliferation, invasion and migration, and intensified the sensitivity of A2780/DDP cells to cisplatin (DDP). GTW, especially GTW+DDP, significantly inhibited the expression of N-cadherin, integrin-linked kinase (ILK), phospho-protein kinase B/AKT (PKB/p-AKT), phospho-glycogen synthase kinase (p-GSK3ß) and Slug, while it increased E-cadherin levels by inhibiting EMT via the ILK/AKT/GSK3ß/Slug signalling pathway. Animal results indicated that GTW, especially GTW+DDP, significantly reduced tumour burden, prolonged the life span of mice, and down-regulated the levels of tumour markers CA125 and HE4 by regulating EMT through the ILK/AKT/GSK3ß/Slug signalling pathway. Conclusion: Our results highlighted the significance of EMT in EOC metastasis, invasiveness and resistance to DDP and investigated the potential role of GTW as an adjuvant therapeutic agent in chemo-resistant EOC.

Probiotic Supplement Preparation Relieves Test Anxiety by Regulating Intestinal Microbiota in College Students.

Test anxiety creates barriers to learning and performance, which further affects students' social, behavioural, and emotional development. Currently, the medication to treat test anxiety has not been reported yet. Here, we enrolled 120 students to evaluate the effect of probiotic supplement preparation (PSP) on test anxiety from the aspect of the intestinal microbiota. We found that the intake of PSP alleviated the symptoms of depression and anxiety in students with test anxiety by evaluating their mental state using the Hamilton Depression Rating Scale and Hamilton Anxiety Scale. High-throughput sequencing results indicated that the consumption of PSP increased the abundance of Streptococcus and Akkermansia that was lowered by the anxiety state in the intestinal microbiota of students. Meanwhile, taking PSP reduced the level of intestinal pathogens of Fusobacterium and Clostridium as well. In conclusion, our work shows that PSP can reduce test anxiety and restore the disturbed microbiota to the standard level in Chinese college students, rendering the use of PSP a promising strategy for test anxiety.

Dysbiosis of the rat vagina is efficiently rescued by vaginal microbiota transplantation or probiotic combination.

Vaginal dysbiosis is characterised by a disturbed vaginal microbiota and is associated with various gynaecological diseases. Owing to its high recurrence rate, there is an urgent need for the development of effective therapeutic agents. In the present study, a vaginal dysbiosis model was developed to study the effect of vaginal microbiota transplantation (VMT) or probiotic combination (containing Lactobacillus helveticus, Lactobacillus crispatus, Lactobacillus acidophilus, Lactobacillus gasseri and Lactobacillus salivarius) on vaginal dysbiosis. Our results indicated that VMT or probiotic combination significantly reduced bacterial-induced inflammation (infiltration of neutrophils, lymphocytes and monocytes) in the uterine wall and the enrichment of pro-inflammatory cytokines [interleukin-1ß (IL-1ß) and tumour necrosis factor-alpha (TNFα)] in vaginal tissue, and restored the disturbed vaginal microbiota to normal levels (increased numbers of Lactobacillus and decreased numbers of Enterobacter and Enterococcus), thus it should be beneficial for avoiding the recurrence of vaginal dysbiosis. Therefore, VMT or probiotic combination might be an effective agent for the treatment of bacterial-induced vaginosis.

Neuroprotective Effect of Ceftriaxone on MPTP-Induced Parkinson's Disease Mouse Model by Regulating Inflammation and Intestinal Microbiota.

Parkinson's disease (PD) is a common degenerative disease of the central nervous system. Although some drugs can alleviate the progress of PD, their long-term use will lead to complications, so it is still necessary to find new drugs to delay or cure PD effectively. In view of the difficulty in developing new drugs, it is imperative to discover new functions of existing compounds that could be used to treat PD. In this study, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was used to induce PD symptoms in a mouse model. Subsequently, these mice were treated with the antibiotic ceftriaxone. Ceftriaxone alleviated the behavioural and neuropathological changes induced by MPTP, downregulated the expression of glial fibrillary acidic protein (GFAP) and ionised calcium-binding adapter molecule 1 (Iba1) as markers of astroglia and microglia, respectively, and reduced the expression of neuroinflammation-related Toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), and phosphorylated nuclear factor kappa-B (p-NF-κB)/NF-κB in the brain of PD mice. In addition, ceftriaxone reduced the abundance of pathogenic bacteria of the genus Proteus and increased the abundance of probiotic Akkermansia. Finally, ceftriaxone treatment increased the expression of the tight junction proteins zona occludens-1(ZO-1) and occludin in the colon, decreased the expression of the inflammation-related proteins TLR4, MyD88, and NF-κB in the colon, and decreased the serum concentration of the proinflammatory cytokines interleukin-1ß (IL-1ß), IL-6, and tumour necrosis factor-α (TNF-α). These results indicate that ceftriaxone had a neuroprotective effect on MPTP-induced PD mice, and its neuroprotective effect could be through regulating inflammation and intestinal microbiota. While we showed that ceftriaxone exerts a neuroprotective effect in an MPTP-induced PD mouse model, our findings are limited to the short-term effects of ceftriaxone. Additional work using transgenic mice is required to determine the long-term effects of ceftriaxone. In addition, the dose and frequency of ceftriaxone use should be evaluated.

Preparation and Evaluation of the Fully Humanized Monoclonal Antibody GD-mAb Against Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) is the major cause of pulmonary and bronchial inflammation in infants, young children, and immunocompromised adults, but therapeutic options to control RSV are limited. In the present study a single chain antibody against RSV (GD-scFv) was screened using phage display library panning technology and a full-length monoclonal antibody (GD-mAb) was developed from GD-scFv based on the sequence encoding Ig VH and Ig VL. The anti-RSV potential of GD-mAb was evaluated in vitro and in mice. Our results indicated that both GD-scFv (4.25 ± 2 nM) and GD-mAb (3.13 ± 0.89 nM) showed high binding capability and strong binding specificity to GD protein. GD-mAb effectively neutralized RSV and reduced the plaque number in a concentration-dependent manner through a plaque reduction neutralization assay. In mice, GD-mAb lowered the lung index and reduced the lung virus titer in the mouse lung (p < 0.05). Antibody treatment reduced the phosphorylated protein level in pathways of TLR4/NF-κB, MAPKs, and PI3K/Akt (p < 0.05) and correlated with an absence of pro-inflammatory factors TNF-α, IL-1ß, and IL-6 in the mouse lung and serum (p < 0.05). In summary, these data suggest that GD-mAb may be an effective therapeutic agent for the treatment of RSV infections. Importance  Currently, only a few therapeutic options are available to control respiratory RSV in humans. In this study, our group developed a full-length monoclonal antibody (GD-mAb) and reported a high binding specificity of the RSV surface glycoproteins G. Moreover, GD-mAb effectively neutralized RSV in vitro, and significantly lowered the lung index and reduced the lung virus titer in an infected mouse lung, which suggests that GD-mAb may serve as an effective antiviral agent for RSV infection.

Therapeutic effect of herb residue fermentation supernatant on spleen­deficient mice.

To minimize the waste of active ingredients present in herb residues, in the present study, probiotics of Bacillus subtilis, Aspergillus oryzae and Lactobacillus plantarum M3 were selected to reuse herb residues from Jianweixiaoshi tablets, and the therapeutic effects of the herb residue fermentation supernatant were evaluated using a spleen­deficient mouse model. The results of the present study indicated that the fermentation supernatant may effectively improve the immunity of mice, as measured by body weight, spleen and thymus index, and inflammatory cytokines, including interleukin (IL)­2, IL­4 and interferon­Î³. The viable cell count and denaturing gradient gel electrophoresis results indicated that the fermentation supernatant markedly enhanced bacterial diversity and the number of lactobacilli in mouse intestines. Therefore, the combination of the Jianweixiaoshi herb residue and probiotics provided a novel method to reuse herb residues and may in the future contribute to the treatment of spleen deficiency.

Evaluation of the accuracy and sensitivity of high­throughput sequencing technology using known microbiota.

Next generation sequencing provides an excellent platform to explore microbiota in any given environment, and little work is required to evaluate the accuracy and sensitivity of high­throughput sequencing technology. In the present study, a known microbiota containing Escherichia coli, Lactobacillus plantarum, Streptococcus thermophilus, Bifidobacterium bifidum, Bacillus subtilis, Enterococcus faecalis and Salmonella typhimurium was used to evaluate the high­throughput sequencing technology. The results suggested that there were 122.7 operational taxonomic units (OTUs) in all groups, which is 17.5­fold (the whole OTU number/the actual bacterial number) greater compared with the actual microbial number in each group, and the Venn method indicated that only 46.38% (64/138), 58.70% (81/138), 86.13% (118/137), 83.57% (117/140) and 89.29% (125/140) of the common OTUs were identified in groups A, B, C, D and E, of which the majority of OTUs did not belong to known bacteria. In addition, the DNA extraction and amplification efficiency failed to identify bacteria at the phylum, class, order, family, genus and species levels, which may further increase false information of microbial analysis. In conclusion, the present study provided basic datato investigate the potential drawbacks of high­throughput sequencing technology, which will help researchers to avoid exaggerating the bacterial number when this technology is applied to study microbiota in particular environments.