[Research progress on colorectal cancer identification based on convolutional neural network].

Colorectal cancer (CRC) is a common malignant tumor that seriously threatens human health. CRC presents a formidable challenge in terms of accurate identification due to its indistinct boundaries. With the widespread adoption of convolutional neural networks (CNNs) in image processing, leveraging CNNs for automatic classification and segmentation holds immense potential for enhancing the efficiency of colorectal cancer recognition and reducing treatment costs. This paper explores the imperative necessity for applying CNNs in clinical diagnosis of CRC. It provides an elaborate overview on research advancements pertaining to CNNs and their improved models in CRC classification and segmentation. Furthermore, this work summarizes the ideas and common methods for optimizing network performance and discusses the challenges faced by CNNs as well as future development trends in their application towards CRC classification and segmentation, thereby promoting their utilization within clinical diagnosis.

Identification of Rickettsia spp., Anaplasma spp., and an Ehrlichia canis-like agent in Rhipicephalus microplus from Southwest and South-Central China.

Rhipicephalus microplus is considered to be the most important tick infesting cattle, buffalo, horse, goats as well as other animals. They transmit diseases between domestic animals and act as vectors of a variety of zoonotic pathogens. Although pathogens harbored by R. microplus have been extensively studied, the Rickettsiales pathogens vectored by R. microplus in some areas of China remained largely unexplored. From August to October 2020, a total of 291 R. microplus ticks were collected from goats and cattle in three Southern China provinces, Guangxi (n = 138), Sichuan (n = 120) and Hubei (n = 33) provinces. Phylogenetic analysis based on COI gene sequences shows that these ticks are divided into three distinct clades, indicating the remarkable genetic diversity of R. microplus ticks in China. These samples were subsequently screened for the presence of Rickettsia, Anaplasma and Ehrlichia using conventional PCR and sequencing. Subsequently, five bacterial species were identified. Out of the 120 tick DNA samples from Sichuan province, 35.83% (43/120) were positive for Rickettsia sp. belonging to spotted fever group (SFG), 12.50% (15/120) were positive for Anaplasma marginale and 0.83% (1/120) was identified as A. platys. From the 138 DNA samples from Guangxi province, an Ehrlichia canis-like and Rickettsia sp. were detected, with a positive rate of 11.59% (16/138) and 2.17% (3/138), respectively. A. capra DNA was detected in 4 out of 33 (12.12%) samples from Hubei province. Notably, the 16S, gltA and groEL sequences of the E. canis-like are closely related to the E. canis strain previously identified from China, and form a distinct cluster in the phylogenetic trees. Collectively, our results expand the knowledge on tick-borne Rickettsiales pathogens in China. Because the state of engorgement of ticks was not recorded, it is not clear at this stage whether these pathogens are infecting the ticks or are simply present in the blood meal. Given the public health significance of SFG Rickettsia, A. capra, A. platys and E. canis, a thorough investigation of the diversity and presence of pathogens in R. microplus in areas with tick-associated diseases are needed.

Tumor acidic microenvironment-induced drug release of RGD peptide nanoparticles for cellular uptake and cancer therapy.

Spatial accuracy is crucial in drug delivery, especially to increase the efficacy and reduce the side effects of antitumor drugs. In this study, we developed a simple and broadly applicable strategy in which a target peptide ligand was introduced to construct a pH-responsive drug-loading system to achieve targeted delivery and drug release in lesions. In addition to reaching the tumor tissue through passive targeting modalities such as the enhanced permeability and retention (EPR) effect, active targeting nanoparticles used RGD motifs coupled to nanocarriers to specifically bind certain integrins, such as ανß3, which is expressed on the surface of tumor cells, to achieve active tumor cell targeting. Self-assembling peptides have significant advantages in their structural design. The amphiphilic peptide LKR could form a spherical and self-assembled nanoparticle, which encapsulated the fat-soluble antitumor drug doxorubicin (Dox) in neutral medium. The Dox-encapsulating peptide nanoparticles swelled and burst, rapidly releasing Dox in an acidic microenvironment. Flow cytometry and fluorescence detection showed that the self-assembled LKR nanoparticles enhanced the drug accumulation in tumor cells compared with normal mammalian cells. The Dox-encapsulating peptide nanoparticles exhibited desirable antitumor effects in vivo. In summary, the acidic microenvironment of tumors was used to induce drug release from a targeted peptide drug-loading system to enhance cellular uptake and therapeutic effects in situ, providing a promising therapeutic approach for the treatment of major diseases such as hepatoma.

Improving the production of AHL lactonase AiiO-AIO6 from Ochrobactrum sp. M231 in intracellular protease-deficient Bacillus subtilis.

Quorum quenching (QQ) blocks bacterial cell-to-cell communication (i.e., quorum sensing), and is a promising antipathogenic strategy to control bacterial infection via inhibition of virulence factor expression and biofilm formation. QQ enzyme AiiO-AIO6 from Ochrobactrum sp. M231 has several excellent properties and shows biotherapeutic potential against important bacterial pathogens of aquatic species. AiiO-AIO6 can be secretory expressed in Bacillus subtilis via a non-classical secretion pathway. To improve AiiO-AIO6 production, four intracellular protease-deletion mutants of B. subtilis 1A751 were constructed by individually knocking out the intracellular protease-encoding genes (tepA, ymfH, yrrN and ywpE). The AiiO-AIO6 expression plasmid pWB-AIO6BS was transformed into the B. subtilis 1A751 and its four intracellular protease-deletion derivatives. Results showed that all recombinant intracellular protease-deletion derivatives (BSΔtepA, BSΔymfH, BSΔyrrN and BSΔywpE) had a positive impact on AiiO-AIO6 production. The highest amount of AiiO-AIO6 extracellular production of BSΔywpE in shake flask reached 1416.47 U/mL/OD600, which was about 121% higher than that of the wild-type strain. Furthermore, LC-MS/MS analysis of the degrading products of 3-oxo-C8-HSL by purification of AiiO-AIO6 indicated that AiiO-AIO6 was an AHL-lactonase which hydrolyzes the lactone ring of AHLs. Phylogenetic analysis showed that AiiO-AIO6 was classified as a member of the α/ß hydrolase family with a conserved "nucleophile-acid-histidine" catalytic triad. In summary, this study showed that intracellular proteases were responsible for the reduced yields of heterologous proteins and provided an efficient strategy to enhance the extracellular production of AHL lactonase AiiO-AIO6.

Improving extracellular production of Serratia marcescens lytic polysaccharide monooxygenase CBP21 and Aeromonas veronii B565 chitinase Chi92 in Escherichia coli and their synergism.

Lytic polysaccharide monooxygenases (LPMOs) can oxidize recalcitrant polysaccharides and boost the conversion of the second most abundant polysaccharide chitin by chitinase. In this study, we aimed to achieve the efficient extracellular production of Serratia marcescens LPMO CBP21 and Aeromonas veronii B565 chitinase Chi92 by Escherichia coli. Twelve signal peptides reported with high secretion efficiency were screened to assess the extracellular production efficiency of CBP21 and Chi92, with glycine used as a medium supplement. The results showed that PelB was the most productive signal peptide for the extracellular production of CBP21 and Chi92 in E. coli. Furthermore, CBP21 facilitated the degradation of the three chitin substrates (colloidal chitin, ß-chitin, and α-chitin) by Chi92. This study will be valuable for the industrial production and application of the two enzymes for chitin degradation.

Secretory expression of a heterologous protein, Aiio-AIO6BS, in Bacillus subtilis via a non-classical secretion pathway.

The quenching enzyme AIO6 (AiiO-AIO6) has been reported as a feed additive preparation for application in aquaculture and biological control of pathogenic Aeromonas hydrophila. We developed an economical strategy to express AIO6BS (AiiO-AIO6BS, codon optimized AIO6 in Bacillus subtilis) in Bacillus subtilis for facilitating its widespread application. Promoter p43 without the signal peptide was used for secretory expression of AIO6BS in B. subtilis. Western blotting analysis demonstrated that AIO6BS was successfully expressed and secreted into the cell culture. Expression analysis of AIO6BS in the single or double mutant of the lytC and lytD genes for cell autolysis in B. subtilis 1A751 and cell autolysis-resistant engineered strain LM2531 derived from the wild type 168 indicated that the release of the heterologous protein AIO6BS was not simply mediated by cell lysis. Expression level of AIO6BS did not change in the mutants of B. subtilis that harbored mutations in the secA, tatAC, or ecsA genes compared with that in the parent wild type strain. These results suggested the AIO6BS protein was likely secreted via a non-classical secretion pathway. The expression analysis of the various N- or C-terminal truncated gene products indicated that AIO6BS probably acts as an export signal to direct its self-secretion across the cell membrane.

Involvement of SpoVG in hemolysis caused by Bacillus subtilis.

Bacillus subtilis is a facultative anaerobic Gram-positive non-pathogenic bacterium that includes members displaying hemolytic activity. To identify the genes responsible for hemolysis, a random mariner-based transposon insertion mutant library of B. subtilis 168 was constructed. More than 20,000 colonies were screened for the hypohemolytic phenotype on blood agar plates. One mutant showed significantly less pronounced hemolytic phenotype than the wild type. DNA sequencing and Southern blot analysis showed this mutant has a single transposable element inserted into the open reading frame (ORF) of the spoVG gene; complementation of the spoVG-disrupted mutant with a wild-type copy restored its hemolytic phenotype. It was therefore concluded that the spoVG gene, which plays a role in regulating asymmetric septation during sporulation in B. subtilis, is involved in hemolysis by B. subtilis.

[Transiently secretory expression of DsRed driven by fibroin heavy chain promoter of Bombyx mori].

Based on the character of strong promoter of the fibroin gene and high level secretion of fibroin of Bombyx mori, we amplified the promoter of heavy chain gene (Fib-H) and its downstream signal peptide sequence (FibHS) by PCR. After that, we cloned the PCR product in pBluescriptII SK (+) to form the vector pSK-FibHS and analyzed its sequence. The sequence identity was 99% comparable to that of the reported sequence by Blast on line. Then we digested pSk-Ser-DsRed-PolyA with Sal IKpn I to get DsRed-PolyA DNA fragment and subcloned it into vector pSK-FibHS to generate a transitorily secretory expression vector pSK-FibHS-DsRed-PolyA. After identified the recombinant plasmid by restriction enzyme digestion, we transfected pSK-FibHS-DsRed-PolyA into BmN cells by liposome. From the cells transfected with the recombinant vector, what the red fluorescence could be detected verified that the recombinant vector could express DsRed in BmN cells transiently. Furthermore, when silkworm had been injected with the recombinant vector pSK-FibHS-DsRed-PolyA, red fluorescence could be observed in the lumen of silk gland of silkworm. The result indicated that DsRed expressed transiently and was secreted into lumen of the silk gland. Therefore, we supposed that the cloned sequence (FibHS) possessed signal peptide bio-function. Moreover, this study would lay a foundation for the research on secretory expression of exogenous gene by silk gland bioreactor.