An ultrasensitive ELISA to assay femtomolar level SARS-CoV-2 antigen based on specific peptide and tyramine signal amplification.

The SARS-CoV-2 spreading rapidly has aroused catastrophic public healthcare issues and economy crisis worldwide. It plays predominant role to rapidly and accurately diagnose the virus for effective prevention and treatment. As an abundant transmembrane protein, spike protein (SP) is one of the most valuable antigenic biomarkers for diagnosis of COVID-19. Herein a phage expression of WNLDLSQWLPPM peptide specific to SARS-CoV-2 SP was screened. Molecular docking revealed that the isolated peptide binds to major antigenic epitope locating at S2 subunit with hydrogen bonding. Taking the specific peptide as antigen sensing probe and tyramine signal amplification (TSA), an ultrasensitive "peptide-antigen-antibody" ELISA (p-ELISA) was explored, by which the limit of detection (LOD) was 14 fM and 2.8 fM SARS-CoV-2 SP antigen for first TSA and secondary TSA, respectively. Compared with the LOD by the p-ELISA by direct mode, the sensitivity with 2nd TSA enhanced 100 times. Further, the proposed p-ELISA method can detect SARS-CoV-2 pseudoviruses down to 10 and 3 TCID50/mL spiked in healthy nasal swab sample with 1st TSA and 2nd TSA, separately. Thus, the proposed p-ELISA method with TSA is expected to be a promising ultrasensitive tool for rapidly detecting SARS-CoV-2 antigen to help control the infectious disease.

Gut microbiota and colorectal cancer metastasis.

Gut microbiota play critical roles in the development of colorectal cancer (CRC) metastasis, but the underlying mechanisms remain elusive. This review discusses the molecular mechanisms by which the gut microbiota contribute to a tumor-permissive microenvironment and facilitate malignant transformation and dissemination of tumor cells, thereby mediating CRC metastasis.

A High Hepatic Uptake of Conjugated Bile Acids Promotes Colorectal Cancer-Associated Liver Metastasis.

The liver is the most common site for colorectal cancer (CRC)-associated metastasis. There remain unsatisfactory medications in liver metastasis given the incomplete understanding of pathogenic mechanisms. Herein, with an orthotopic implantation model fed either regular or high-fat diets (HFD), more liver metastases were associated with an expansion of conjugated bile acids (BAs), particularly taurocholic acid (TCA) in the liver, and an increased gene expression of Na+-taurocholate cotransporting polypeptide (NTCP). Such hepatic BA change was more apparently shown in the HFD group. In the same model, TCA was proven to promote liver metastases and induce a tumor-favorable microenvironment in the liver, characterizing a high level of fibroblast activation and increased proportions of myeloid-derived immune cells. Hepatic stellate cells, a liver-residing source of fibroblasts, were dose-dependently activated by TCA, and their conditioned medium significantly enhanced the migration capability of CRC cells. Blocking hepatic BA uptake with NTCP neutralized antibody can effectively repress TCA-triggered liver metastases, with an evident suppression of tumor microenvironment niche formation. This study points to a new BA-driven mechanism of CRC-associated liver metastases, suggesting that a reduction of TCA overexposure by limiting liver uptake is a potential therapeutic option for CRC-associated liver metastasis.

Effects of gut microbiota on immune responses and immunotherapy in colorectal cancer.

Accumulating evidence suggests that gut microbial dysbiosis is implicated in colorectal cancer (CRC) initiation and progression through interaction with host immune system. Given the intimate relationship between the gut microbiota and the antitumor immune responses, the microbiota has proven to be effective targets in modulating immunotherapy responses of preclinical CRC models. However, the proposed putative mechanisms of how these bacteria affect immune responses and immunotherapy efficacy remains obscure. In this review, we summarize recent findings of clinical gut microbial dysbiosis in CRC patients, the reciprocal interactions between gut microbiota and the innate and/or the adaptive immune system, as well as the effect of gut microbiota on immunotherapy response in CRC. Increased understanding of the gut microbiota-immune system interactions will benefit the rational application of microbiota to the clinical promising biomarker or therapeutic strategy as a cancer immunotherapy adjuvant.

Sensitive electrochemical sequential enzyme biosensor for glucose and starch based on glucoamylase- and glucose oxidase-controllably co-displayed yeast recombinant.

The sequential enzyme biosensors hold significant importance in measuring species which are usually hard to process with single-enzyme-based biosensors. However, sequential enzyme electrodes experience critical issues such as low catalytic efficiency, insensitivity and poor reproducibility. In this work, yeast surface co-displaying sequential enzymes of glucoamylase (GA) and glucose oxidase (GOx) with controllable ratios through the specific cohesion-dockerin protein interaction was explored, by which starch hydrolyzing by GA into glucose is the rate-limiting step. The modified electrodes were prepared by immobilizing yeast-GA&GOx whole-cell and reduced graphene oxide (RGO) on glassy carbon electrode (GCE), for which the direct electron transfer between the electrode and recombinant GOx was arrived. Interestingly, the current responses of sensors to starch and glucose are dependent on the displayed enzyme composition, of which the yeast-GA&GOx (2:1) exhibited the highest current. Thereafter, sequential enzyme sensor of yeast-GA&GOx (2:1)/RGO/GCE was developed. Based on reduction detection at negative potential without interference, the sensor is stable and capable of assaying glucose (linear range: 2.0-100 mg/L) or starch (linear range, 50-3500 mg/L), separately. Coupled with yeast-GOx/RGO/GCE glucose sensor, both glucose and starch in real samples can be detected satisfactorily. This work provides new ideas for the development of other sequential enzyme electrodes for potential applications.

An ultrasensitive label-free electrochemical impedimetric immunosensor for vascular endothelial growth factor based on specific phage via negative pre-screening.

As a vascular growth regulator, vascular endothelial growth factor (VEGF) exerts significant biological roles through specific binding to its receptors on the vascular endothelial cells. VEGF165 is generally referenced as a potential therapeutic target of many malignant tumors. In this study, a negative pre-screening strategy with structurally analogous members of VEGF121, VEGFC and VEGFD was first proposed for VEGF165 biopanning, aiming at significantly improving the specificity of the selected phage monoclones. Indirect ELISA experiment showed that the phage monoclone expressing peptide SPFLLRM demonstrates excellent affinity and specificity. Then a VEGF165 electrochemical impedimetric spectroscopy (EIS) immunosensor was constructed by above specific phage modified electrode. After optimizing the experimental conditions, the as-explored EIS immunosensor had a linear range of 0.5-1000 pg/mL with the limit of detection of 0.15 pg/mL VEGF165. In addition, the developed phage-based EIS immunosensor was applied to satisfactorily detect VEGF165 in human serum samples. Considering its ultra-sensitivity, good selectivity, batch reproducibility and stability, the screened selective phage-based EIS sensor is envisioned potential application in diagnosis and therapy.

A membraneless starch/O2 biofuel cell based on bacterial surface regulable displayed sequential enzymes of glucoamylase and glucose dehydrogenase.

Enzymatic biofuel cells (EBFCs) provide a new strategy to enable direct biomass-to-electricity conversion, posing considerable demand on sequential enzymes. However, artificial blend of multi-enzyme systems often suffer biocatalytic inefficiency due to the rambling mixture of catalytic units. In an attempt to construct a high-performance starch/O2 EBFC, herein we prepared a starch-oxidizing bioanode based on displaying a sequential enzyme system of glucoamylase (GA) and glucose dehydrogenase (GDH) on E.coli cell surfaces in a precise way using cohesin-dockerin interactions. The enzyme stoichiometry was optimized, with GA&GDH (3:1)-E.coli exhibiting the highest catalytic reaction rate. The bioanode employed polymerized methylene blue (polyMB) to collect electrons from the oxidation of NADH into NAD+, which jointly oxidized starch together with co-displayed GA and GDH. The bioanode was oxygen-insensitive, which can be combined with a laccase based biocathode, resulting in a membranless starch/O2 EBFC in a non-compartmentalized configuration. The optimal EBFC exhibited an open-circuit voltage (OCV) of 0.74 V, a maximum power density of 30.1 ± 2.8 µW cm-2, and good operational stability.

Hierarchical porous MoS2 particles: excellent multi-enzyme-like activities, mechanism and its sensitive phenol sensing based on inhibition of sulfite oxidase mimics.

It is important to exploit highly efficient methods for detecting pollutants selectively and sensitively. Artificial enzymes are promising to replace natural enzymes with diverse functions for sustainable developments and various applications. However, it remains the challenge to develop novel mimic enzymes or multi-enzyme mimics for pollutant detection. Herein we report hierarchical porous MoS2 particles prepared by a simple hydrothermal method, which demonstrated excellent sulfite oxidase (SuOx)-, nicotinamide adenine dinucleotide (NADH) oxidase- and superoxide dismutase-mimicking activities. In addition, the catalytic conditions for SuOx-like and NADH oxidase-like activities of MoS2 were optimized. The catalytic mechanism of the NADH oxidase mimics is that O2 involves in the oxidation of NADH, to generate O2.- intermediate and finally turn to H2O2, while SuOx mimics comes from that MoS2 particles can effectively catalyze sulfite to reduce [Fe(CN)6]3-. Based on the excellent SuOx-like activity of MoS2 particles, while phenol can inhibit the oxidation of sulfite, a phenol colorimetric sensor was explored with the dynamic range of 2-1000 µM and the limit of detection of 0.72 µM, applicable to detect phenol in effluents. Therefore, MoS2 particles with the SuOx-like, NADH oxidase-like and SOD-like activities has broad application prospects in environmental monitoring and bio-analysis.

Capsaicin suppressed activity of prostate cancer stem cells by inhibition of Wnt/ß-catenin pathway.

Cancer stem cells (CSCs) are considered as the origin and driving cells of cancer, and play a key role in the progress of cancer. Studies have shown that capsaicin exerted inhibitory effect on prostate cancer cells, however, the effects of capsaicin on prostate CSCs remain undefined. In the present study, we showed that capsaicin could downregulate prostate CSCs markers and inhibit the growth of PC-3 and DU145 prostate cancer stem cells. Further, we found capsaicin suppressed the expression of Wnt-2, p-GSK3ß and ß-catenin, along with downregulation of Wnt/ß-catenin pathway target genes c-myc and cyclinD1. Using LiCl, a activator of Wnt/ß-catenin pathway, we found activation of Wnt/ß-catenin pathway could ameliorate the downregulation of prostate CSCs markers and the growth inhibition induced by capsaicin in prostate cancer stem cells. Those data suggested that the inhibition effect of capsaicin on prostate cancer stem cells and the anti-cancer effect of capsaicin on prostate cancer stem cell may be mediated by Wnt/ß-catenin pathway. Findings from this study reveals for the first time the potential role and mechanisms of capsaicin on prostate cancer stem cells.

Modulation of miR-34a in curcumin-induced antiproliferation of prostate cancer cells.

Curcumin is a phytochemical which exhibits significant inhibitory effect in multiple cancers including prostate cancer. MicroRNA-34a (miR-34a) was found to be a master tumor suppressor miRNA and regulated the growth of cancer cells. To date, however, the role of miR-34a in the anticancer action of curcumin against prostate cancer has been rarely reported. In the present study, we showed that curcumin altered the expression of cell cycle-related genes (cyclin D1, PCNA, and p21) and inhibited the proliferation of prostate cancer cells. Furthermore, we found that curcumin significantly upregulated the expression of miR-34a, along with the downregulated expression of ß-catenin and c-myc in three prostate cancer cell lines. Inhibition of miR-34a activated ß-catenin/c-myc axis, altered cell cycle-related genes expression and significantly suppressed the antiproliferation effect of curcumin in prostate cancer cells. Findings from this study revealed that miR-34a plays an important role in the antiproliferation effect of curcumin in prostate cancer.

A Rare Case of Unilateral Isolated Naso-Ocular Cleft Treated by a Rotation-Advancement Technique.

The isolated naso-ocular cleft is rare. In this article, the authors report an extremely rare case of unilateral isolated naso-ocular cleft. And a new rotation-advancement technique was used in repair. Result was satisfied, especially the esthetical scar location. But if an upper lid flap was performed simultaneously, the result should be better.

Congenital Midline Sinus of the Upper Lip.

An extremely rare patient of congenital midline sinus of the upper lip in a 2-year-old boy is presented. The sinus was located on the midline of the philtrum just below the base of the columella. Surgical removal of the sinus was performed via an extra- and inraoral approach. Histopathological examination of the resected sinus revealed it to be lined by keratinized stratified squamous epithelium with sebaceous glands and hair follicles. Three postulates can account for the formation of the congenital midline sinus of the upper lip: the fusion theory, the merging theory, and the invagination theory. However, the etiology of this rare abnormality remains obscure.

Two rare cases of simultaneous Tessier number 3 cleft, contralateral cleft lip, and signs of amniotic band syndrome.

The Tessier number 3 cleft is rare. In this paper, we report two extremely rare cases of simultaneous Tessier number 3 cleft, contralateral cleft lip, and signs of amniotic band syndrome. In the two cases, we confirmed that amniotic bands were the probable cause of the Tessier number 3 cleft, where swallowed fibrous strands of amniotic bands entangle a typical cleft lip and cause the more severe Tessier number 3 cleft. In this study, Z-plasty was performed for one case, and a straight-line method was used for the other. Postoperatively, the appearance of both patients was satisfactory, as expected. Consequently, treatment for the Tessier number 3 cleft should be designed individually based on the severity of deformity.

Spatial and temporal expression of c-Kit in the development of the murine submandibular gland.

The c-Kit pathway is important in the development of many mammalian cells and organs and is indispensable for the development of hematopoiesis, melanocytes, and primordial germ cells. Loss-of-function mutations in c-Kit lead to perinatal death in mouse embryos. Previously, c-Kit has been used as one of salivary epithelial stem or progenitor cell markers in mouse, its specific temporo-spatial expression pattern and function in developing murine submandibular gland (SMG) is still unclear. Here we used quantitative real-time PCR, in situ hybridization, and immunohistochemistry analysis to detect c-Kit expression during the development of the murine SMG. We found that c-Kit was expressed in the epithelia of developing SMGs from embryonic day 11.5 (E11.5; initial bud stage) to postnatal day 90 (P90; when the SMG is completely mature). c-Kit expression in the end bud epithelium increased during prenatal development and then gradually decreased after birth until its expression was undetectable in mature acini at P30. Moreover, c-Kit was expressed in the SMG primordial cord at the initial bud, pseudoglandular, canacular, and terminal end bud stages. c-Kit was also expressed in the presumptive ductal cells adjacent to the developing acini. By the late terminal end bud stage on P14, c-Kit expression could not be detected in ductal cells. However, c-Kit expression was detected in ductal cells at P30, and its expression had increased dramatically at P90. Taken together, these findings describe the spatial and temporal expression pattern of c-Kit in the developing murine SMG and suggest that c-Kit may play roles in epithelial histo-morphogenesis and in ductal progenitor cell homeostasis in the SMG.

Allogeneic mesenchymal stem cell therapy for bisphosphonate-related jaw osteonecrosis in Swine.

Bisphosphonates (BPs), which are used to treat a variety of clinical disorders, have the side effect of jawbone necrosis. Currently, there is no reliable treatment for BP-related osteonecrosis of the jaw (BRONJ) due to a lack of understanding of its pathogenesis. To investigate the pathogenesis of BRONJ and observe the treatment effect of bone marrow mesenchymal stem cell (BMMSC) transplantation, we established a preclinical animal model of BRONJ in miniature pigs (minipigs). After treatment with zoledronic acid, the clinical and radiographic manifestations of BRONJ could be observed in minipigs after first premolar extraction. The biological and immunological properties of BMMSCs were impaired in the BP-treated minipigs. Moreover, the ratio of Foxp3-positive regulatory T-cells (Tregs) in peripheral blood decreased, and interleukin (IL)-17 increased in the serum of BP-treated minipigs. After allogeneic BMMSC transplantation via intravenous infusion, mucosal healing and bone reconstruction were observed; IL-17 levels were reduced; and Tregs were elevated. In summary, we established a clinically relevant BRONJ model in minipigs and tested a promising allogeneic BMMSC-based therapy, which may have potential clinical applications for treating BRONJ.

Mesenchymal stromal cell-based treatment of jaw osteoradionecrosis in Swine.

Jaw osteoradionecrosis (ORN) is a common and serious complication of radiation therapy for head and neck cancers. Bone marrow mesenchymal stromal cells (BMMSCs) are multipotent postnatal stem cells and have been widely used in clinical therapies. In the present study, we generated the mandibular ORN model in swine using a combination of single-dose 25-Gy irradiation and tooth extraction. A typical ORN phenotype, including loss of bone regeneration capacity and collagen collapse with the obliteration of vessels, gradually appeared after irradiation. After autologous BMMSC transplantation, new bone and vessels were regenerated, and the advanced mandibular ORN was treated successfully. In summary, we developed a swine model of jaw ORN, and our results indicate that autologous BMMSC transplantation may be a promising therapeutic approach for ORN.

[The influences on mandibular development after removing the outer cortex of mandibular body in childhood minitype pigs].

OBJECTIVE: To study the influences on mandibular development after removing the outer cortex of mandibular body in childhood minitype pig. METHODS: Six childhood minitype pigs were selected as the experimental animals. The outer cortex of mandibular body measured as 3.0 cm x 1.5 cm was removed in one side, and the other side remained intact as the control. The changes of mandibular modality and occlusion relationship as well as the histological and biomechanical changes were studied 24 weeks after operation. RESULTS: There was no obvious difference compared with the control side in the height of the mandibular ramus and the length of the mandibular body, However, lateral deviation occlusion was found in some animals. The body thickness was thinner than that of the control side, there were no obvious biomechanical and histological differences between the two sides. CONCLUSIONS: There was less influence on the growth of mandibular bone after removing one side of the outer cortex of the mandibular body in childhood minitype pig. But further study should be done for the cause of the lateral deviation of the mandible in part of the animals.

[The surgical management of unilateral Craniofacial Atrophy and Hypoplasia].

OBJECTIVE: To study the surgical reconstruction of unilateral Craniofacial Atrophy and Hypoplasia. METHODS: According to the etiological factors and severity of the facial deformities, different methods are employed, including bone framework reconstruction, soft tissue transplantation, orthognathic surgery. RESULTS: From September 1998 to August 2004, 42 cases were treated, Include: Hemifacial Microsomia 22 cases, Hemifacial Atrophy 16 cases, unilateral facial hypoplasia due to radiation 4 cases. Miniplate and transplants extrusion occurred on 2 post radiation patients due to poor soft tissue coverage, infection occurred on 1 patient after mandibular ramus reconstruction using autogenous rib and contralateral mandibular outer cortex. The leaving patients recovered well and the facial asymmetry were improved greatly. CONCLUSIONS: Facial asymmetry due to unilateral Craniofacial Atrophy or Hypoplasia is a common and complex condition for surgical management, The surgical plan should be delicated made individually according to the severity of the soft tissue and the underlying bone framework.

Heterogeneity in predisposition of hepatic cells to be induced into pancreatic endocrine cells by PDX-1.

AIM: The role of Pancreatic and Duodenal Homeobox-1 (PDX-1) as a major regulator of pancreatic development determines the function and phenotype of beta cell. In this study, potential plasticity of liver cells into pancreatic endocrine cells induced by PDX-1 was evaluated. METHODS: Human hepatoma cell line HepG2 was stably transfected with mammalian expression plasmid pcDNA3-PDX encoding human PDX-1 gene. Ectopic expression of PDX-1 and insulin were detected by RT-PCR, Western blot and/or immunostaining. PDX-1(+) HepG2 cells were transplanted under renal capsule of STZ-induced diabetic nude mice (n = 16) to examine the inducing effect in vivo. RESULTS: Exogenous PDX-1 transgene was proved to express effectively in HepG2 cell at both mRNA and protein levels. The expression of endogenous insulin and some beta cell-specific differentiation markers and transcription factors were not induced in PDX-1(+) HepG2 cells. When transplanted under renal capsule of STZ-induced diabetic nude mice, PDX-1(+) HepG2 cells did not generate insulin-producing cells. These data indicated that stable transfected PDX-1 could not convert hepatoma cell line HepG2 to pancreatic cells in vitro or in vivo. Mature hepatocytes might need much more complicated or rigorous conditions to be shifted to insulin-producing cells. CONCLUSION: The expression of exogenous PDX-1 is not sufficient to induce relatively mature hepatocytes differentiating into insulin-producing cells.

[Expression of nestin and neurogenin 3 in the human fetal pancreas].

OBJECTIVE: To examine the expression of nestin and neurogenin 3 (Ngn3), the markers of pancreatic stem cells, in the human fetal pancreas. METHODS: The human fetal pancreas tissue of 12 and 14 weeks were examined for the expression of nestin and Ngn3 using the techniques of immunofluorescence dye and RT-PCR. RESULTS: Both nestin and Ngn3 expressed widely in 12 and 14 weeks before in human fetal pancreatic tissue. In these positive cells there was no co-expressing insulin or glucagon. There were nestin and Ngn3 co-expressing cells in ducts but not in the islets. The results of RT-PCR also indicated the expression of nestin and Ngn3. CONCLUSIONS: There was no expression of the markers of mature endocrine cells in the nestin and Ngn3 positive cells, and they were the marks of no-differentiation cells in the human fetal pancreatic tissue.