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Abstract Background Preventing post-weaning diarrhea (PWD) in weaned piglets is a crucial challenge in the swine production industry. The stress of weaning, dietary shifts from maternal milk to solid feed, and environmental changes lead to decreased microbial diversity, increased pathogen abundance, and compromised intestinal integrity. We have previously identified Lactiplantibacillus argentoratensis AGMB00912 (LA) in healthy porcine feces, which demonstrated antimicrobial activity against pathogens and enhanced short-chain fatty acid production. This research aimed to evaluate the efficacy of LA strain supplementation as a strategy to inhibit PWD and enhance overall growth performance in weaned piglets. Results LA supplementation in weaned piglets significantly increased body weight gain, average daily gain, and average daily feed intake. It also alleviated diarrhea symptoms (diarrhea score and incidence). Notably, LA was found to enrich beneficial microbial populations (Lactobacillus, Anaerobutyricum, Roseburia, Lachnospiraceae, and Blautia) while reducing the abundance of harmful bacteria (Helicobacter and Campylobacter). This not only reduces the direct impact of pathogens but also improves the overall gut microbiota structure, thus enhancing the resilience of weaned piglets. LA treatment also promotes the growth of the small intestinal epithelial structure, strengthens gut barrier integrity, and increases short-chain fatty acid levels in the gut. Conclusions The study findings demonstrate the promising potential of LA in preventing PWD. Supplementation with the LA strain offers a promising feed additive for improving intestinal health and growth in piglets during the weaning transition, with the potential to significantly reduce the incidence and severity of PWD.

Abstract This study aimed to evaluate the efficacy of Lactiplantibacillus argentoratensis AGMB00912 (LA) in reducing Salmonella Typhimurium infection in weaned piglets. The investigation focused on the influence of LA on the gut microbiota composition, growth performance, and Salmonella fecal shedding. The results indicated that LA supplementation significantly improved average daily gain and reduced the prevalence and severity of diarrhea. Fecal analysis revealed reduced Salmonella shedding in the LA-supplemented group. Furthermore, LA notably altered the composition of the gut microbiota, increasing the levels of beneficial Bacillus and decreasing those of harmful Proteobacteria and Spirochaetes. Histopathological examination showed less intestinal damage in LA-treated piglets than in the controls. The study also observed that LA affected metabolic functions related to carbohydrate, amino acid, and fatty acid metabolism, thereby enhancing gut health and resilience against infection. Short-chain fatty acid concentrations in the feces were higher in the LA group, suggesting improved gut microbial activity. LA supplementation enriched the population of beneficial bacteria, including Streptococcus, Clostridium, and Bifidobacterium, while reducing the number of harmful bacteria, such as Escherichia and Campylobacter. These findings indicate the potential of LA as a probiotic alternative for swine nutrition, offering protective effects to the gut microbiota against Salmonella infection.

Maintaining a healthy intestinal environment, optimal epithelial barrier integrity, and balanced gut microbiota composition are essential for the growth performance of weaning pigs. We identified Lactiplantibacillus argentoratensis AGMB00912 (LA) in healthy porcine feces as having antimicrobial activity against pathogens and enhanced short-chain fatty acid (SCFA) production. Herein, we assess the protective role of LA using a weaning mouse model with enterotoxigenic Escherichia coli (ETEC) infection. LA treatment improves feed intake and weight gain and alleviates colon shortening. Furthermore, LA inhibits intestinal damage, increases the small intestine villus height compared with the ETEC group, and enhances SCFA production. Using the Kyoto Encyclopedia of Genes and Genomes and other bioinformatic tools, including InterProScan and COGNIZER, we validated the presence of SCFA-producing pathways of LA and Lactiplantibacillus after whole genome sequencing. LA mitigates ETEC-induced shifts in the gut microbiota, decreasing the proportion of Escherichia and Enterococcus and increasing SCFA-producing bacteria, including Kineothrix, Lachnoclostridium, Roseuburia, Lacrimispora, Jutongia, and Blautia. Metabolic functional prediction analysis revealed enhanced functions linked to carbohydrate, amino acid, and vitamin biosynthesis, along with decreased functions associated with infectious bacterial diseases compared to the ETEC group. LA mitigates the adverse effects of ETEC infection in weaning mice, enhances growth performance and intestinal integrity, rebalances gut microbiota, and promotes beneficial metabolic functions. These findings validate the functionality of LA in a small animal model, supporting its potential application in improving the health and growth performance of weaning pigs.

Abstract Background Ticks are ectoparasites capable of directly damaging their hosts and transmitting vector-borne diseases. The ixodid tick Haemaphysalis flava has a broad distribution that extends from East to South Asia. This tick is a reservoir of severe fever with thrombocytopenia syndrome virus (SFTSV) that causes severe hemorrhagic disease, with cases reported from China, Japan and South Korea. Recently, the distribution of H. flava in South Korea was found to overlap with the occurrence of SFTSV. Methods This study was undertaken to discover the molecular resources of H. flava female ticks using the Illumina HiSeq 4000 system, the Trinity de novo sequence assembler and annotation against public databases. The locally curated Protostome database (PANM-DB) was used to screen the putative adaptation-related transcripts classified to gene families, such as angiotensin-converting enzyme, aquaporin, adenylate cyclase, AMP-activated protein kinase, glutamate receptors, heat shock proteins, molecular chaperones, insulin receptor, mitogen-activated protein kinase and solute carrier family proteins. Also, the repeats and simple sequence repeats (SSRs) were screened from the unigenes using RepeatMasker (v4.0.6) and MISA (v1.0) software tools, followed by the designing of SSRs flanking primers using BatchPrimer 3 (v1.0) software. Results The transcriptome produced a total of 69,822 unigenes, of which 46,175 annotated to the homologous proteins in the PANM-DB. The unigenes were also mapped to the EuKaryotic Orthologous Groups (KOG), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) specializations. Promiscuous presence of protein kinase, zinc finger (C2H2-type), reverse transcriptase, and RNA recognition motif domains was observed in the unigenes. A total of 3480 SSRs were screened, of which 1907 and 1274 were found as tri- and dinucleotide repeats, respectively. A list of primer sequences flanking the SSR motifs was detailed for validation of polymorphism in H. flava and the related tick species. Conclusions The reference transcriptome information on H. flava female ticks will be useful for an enriched understanding of tick biology, its competency to act as a vector and the study of species diversity related to disease transmission. Graphical abstract

This study aimed to determine the alleviating effect of broccoli grown with deep sea water mineral (DSWM) fertilizer extracted from deep sea water on the development of colorectal cancer in C57BL/6N mice treated with AOM/DSS. Naturaldream Fertilizer Broccoli (NFB) cultured with deep sea water minerals (DSWM) showed a higher antioxidant effect and mineral content. In addition, orally administered NFB, showed a level of recovery in the colon and spleen tissues of mice compared with those in normal mice through hematoxylin and eosin (H&E) staining. Orally administered NFB showed the inhibition of the expression of inflammatory cytokine factors IL-1β, IL-6, TNF, IFN-γ, and IL-12 while increasing the expression of IL-10. Furthermore, the expression of inflammatory cytokines and NF-κB in the liver tissue was inhibited, and that of inflammatory enzymes, such as COX-2 and iNOS, was reduced. In the colon tissue, the expression of p53 and p21 associated with cell cycle arrest increased, and that of Bcl-2 associated with apoptosis decreased. Additionally, the expression of Bax, Bad, Bim, Bak, caspase 9, and caspase 3 increased, indicating enhanced activation of apoptosis-related factors. These results demonstrate that oral administration of broccoli cultivated using DSWM significantly restores spleen and colon tissues and simultaneously inhibits the NF-κB pathway while significantly decreasing cytokine expression. Moreover, by inducing cell cycle arrest and activating cell apoptosis, they also suggest alleviating AOM/DSS-induced colon cancer symptoms in C57BL/6N mice.

ABSTRACT The gut microbiota (GM) is associated with colorectal cancer (CRC) development. However, studies demonstrating the role of GM in CRC are limited to metagenomic analyses. These studies lack direct evidence proving that the candidate strains are involved in CRC, and isolated probiotics for bacteriotherapy. Therefore, to identify novel GM with anti-CRC activity, we previously isolated gut bacteria from the feces of healthy individuals, screened the isolated GM’s anti-CRC activity, and discovered that cell-free supernatants of GM isolates demonstrated antiproliferative activity against CRC cells. Here, our study identified one of them as Eubacterium callanderi and chose it for further study because the genus Eubacterium has been suggested to contribute to various aspects of gut health; however, the functions are unknown. First, we confirmed that E. callanderi cell-free supernatant (EcCFS) exerted antiproliferative activity—by inducing apoptosis and cell cycle arrest—that was dose-dependent and specific to cancer cell lines. Next, we discovered that EcCFS active molecules were heat stable and protease insensitive. High-performance liquid chromatography analysis revealed that EcCFS contained high butyrate concentrations possessing anticancer activity. Additionally, gas chromatography-mass spectrometry analysis of the aqueous phase of ethyl acetate-extracted EcCFS and an antiproliferation assay of the aqueous phase and 4-aminobutanoic acid (GABA) suggested that GABA is a possible anti-CRC agent. Finally, in the CT26 allograft mouse model, E. callanderi oral administration and EcCFS peri-tumoral injection inhibited tumor growth in vivo. Therefore, our study reveals that E. callanderi has an anti-CRC effect and suggests that it may be a potential candidate for developing probiotics to control CRC. IMPORTANCE The gut microbiota has been reported to be involved in colorectal cancer, as suggested by metagenomic analysis. However, metagenomic analysis has limitations, such as bias in the analysis and the absence of bacterial resources for follow-up studies. Therefore, we attempted to discover gut microorganisms that are related to colorectal cancer using the culturomics method. In this study, we discovered that Eubacterium callanderi possesses anti-colorectal cancer activity in vitro and in vivo, suggesting that E. callanderi could be used in bacteriotherapy for colorectal cancer treatment.

Akkermansia muciniphila is a commensal bacterium using mucin as its sole carbon and nitrogen source. A. muciniphila is a promising candidate for next-generation probiotics to prevent inflammatory and metabolic disorders, including diabetes and obesity, and to increase the response to cancer immunotherapy. In this study, a comparative pan-genome analysis was conducted to investigate the genomic diversity and evolutionary relationships between complete genomes of 27 A. muciniphila strains, including KGMB strains isolated from healthy Koreans. The analysis showed that A. muciniphila strains formed two clades of group A and B in a phylogenetic tree constructed using 1,219 orthologous single-copy core genes. Interestingly, group A comprised of strains from human feces in Korea, whereas most of group B comprised strains from human feces in Europe and China, and from mouse feces. As group A and B branched, mucin hydrolysis played an important role in the stability of the core genome and drove evolution in the direction of defense against invading pathogens, survival in, and colonization in the mucus layer. In addition, WapA and anSME, which function in competition and post-translational modification of sulfatase, respectively, have been a particularly important selective pressure in the evolution of group A. KGMB strains in group A with anSME gene showed sulfatase activity, but KCTC 15667T in group B without anSME did not. Our findings revealed that KGMB strains evolved to gain an edge in the competition with other gut bacteria by increasing the utilization of sulfated mucin, which will allow it to become highly colonized in the gut environment.

In this study, broccoli grown with deep sea water minerals (DSWMs) confirmed anticancer effects in HT-29 colorectal cancer cells and anti-inflammatory effects in C57BL/6 mouse splenocytes. Natural dream cultured broccoli (NB) grown with DSWMs had elevated sodium (Na) and calcium (Ca) levels and enhanced the expression of p53 and p21, both of which are associated with cell cycle arrest in HT-29 colorectal cancer cells. It also decreased the expression of Bax, Bad, Bim, Bak, caspase-9, and caspase-3. In lipopolysaccharide (LPS)-treated C57BL/6 mouse splenocytes, NB produced little nitric oxide (NO). ELISA research indicated that NB decreased IL-1β, IL-6, TNF-α, IFN-γ, and IL-12 expression while increasing NK cell activity. As a result, broccoli cultivated with deep water minerals has better anticancer and anti-inflammatory properties than conventional and organic farming.

Carrot (Daucus carota) is one of the world’s most significant root vegetables, with various bioactivities. This study aimed to investigate the anticancer activity and anti-inflammatory effects of natural dream cultivation carrot (NC). Natural dream cultivation is a cultivation method based on organic farming incorporating minerals. An MTT assay was used to evaluate the inhibitory rate of carrot samples on HT-29 human colon cancer cells, and qPCR was used to assess the mRNA expression of the cell cycle and apoptosis-related genes in the cancer cells. The nitrite oxide (NO) concentration was determined using the Griess method. The levels of inflammatory cytokines in LPS-induced mouse splenocytes were determined using an enzyme-linked immunosorbent assay, and the activity of NK cells was determined using LDH analysis. The results revealed that NC effectively inhibited cancer cell growth rate. Moreover, NC upregulated the mRNA expression of cell-cycle-arrest-related genes (p53 and p21) and apoptosis-related genes (Bim, Bad, Bax, Bak, caspase-9, and caspase-3) in cancer cells while downregulating the expression of anti-apoptotic genes, Bcl-2 and Bcl-xL. NC inhibited NO production and the release of inflammatory cytokines (TNF-α, IL-6, IL-1β, IFN-γ, and IL-12) in LPS-induced mouse splenocytes. NC also demonstrated the ability to stimulate NK cell activation. This study explored the potential mechanisms underlying carrots’ anticancer and anti-inflammatory properties by investigating their inhibitory effects on cancer cells and regulating the inflammatory response. The innovative mineral-supplemented organic cultivation method, as explored in this study, opens new avenues for harnessing the potential of carrots as a functional food source with promising applications in cancer and inflammation management. This research not only provides insights into the bioactive potential of carrots but also contributes to the future development of novel dietary interventions and therapeutics.

Background/Aims: South Korean soldiers are exposed to similar environmental factors. In this study, we sought to evaluate the gut microbiome of healthy young male soldiers (HYMS) and to identify the primary factors influencing the microbiome composition. Methods: We prospectively collected stool from 100 HYMS and performed next-generation sequencing of the 16S rRNA genes of fecal bacteria. Clinical data, including data relating to the diet, smoking, drinking, and exercise, were collected. Results: The relative abundances of the bacterial phyla Firmicutes, Actinobacteria, Bacteroidetes, and Proteobacteria were 72.3%, 14.5%, 8.9%, and 4.0%, respectively. Fifteen species, most of which belonged to Firmicutes (87%), were detected in all examined subjects. Using cluster analysis, we found that the subjects could be divided into the two enterotypes based on the gut microbiome bacterial composition. Compared with enterotype 2 subjects, subjects classified as enterotype 1 tended to be characterized by higher frequencies of potentially harmful lifestyle habits (current smoker: 55.6% vs 36.6%, p=0.222; heavy drinker: 16.7% vs 3.7%, p=0.120; insufficient physical activity: 27.8% vs 14.6%, p=0.318). We identified a significant difference in the microbiome compositions of current and noncurrent smokers (p=0.008); the former differed from the latter mainly in a relatively lower abundance of Bifidobacterium species and a higher abundance of Negativicutes. Conclusions: A high abundance of Actinobacteria and low abundance of Bacteroidetes were the main features distinguishing the gut microbiomes of HYMS, and current smokers could be differentiated from noncurrent smokers by their lower abundance of Bifidobacterium and higher abundance of Negativicutes.

Abstract Background We applied bibliometric tools to original articles published in the official journal of the Korean Knee Society between 1999 and 2018 to identify their characteristics related to knee surgery and to examine the changes in research trends in the last 20 years. Methods Over a 20-year period, 579 original articles were published in the journal Knee Surgery and Related Research (KSRR). We analyzed the title, keywords, and abstract of the article to analyze the research topics and assigned original articles to seven surgical categories as follows: total knee arthroplasty (TKA), unicompartmental knee arthroplasty (UKA), high tibial osteotomy (HTO), arthroscopy, surgery for cruciate ligament, revision surgery, and other surgery. To analyze the trends in research, we divided the study period into two equal parts of 10 years each, examined the percentage of articles in each decade, and analyzed topic trends using the growth rate. Results Among the original articles, 86 on the topic of non-surgery were excluded, and 493 original articles related surgical research were included. Articles related to surgery accounted for 85.2% of the total original articles published annually. By period, this was 85.6% in the first half and 84.8% in the late half (p = 0.76). A total of 493 original articles related to surgery, with the largest number of TKA-related research at 52.1%. In the study period, the largest increase in the percentage of articles was on the topic of HTO surgery, by 149%. The topics of UKA and revision surgery increased by 95.3% and 33.9%. The topic of TKA increased by 5.9% and the topic of surgery for cruciate ligament decreased by 18.7%. The topic of arthroscopy showed the largest decrease, by 47.6%. Conclusions The bibliometric findings of this study suggest that the majority of surgery-related original articles published in KSRR during the last 20 years involved research about TKA surgery, and the greatest relative increase over the study period involved research about HTO surgery. The authors expect that the analysis of characteristics and research trends of original articles published in KSRR will provide useful information about KSRR for future researchers.

The gut microbiota (GM) has been shown to be closely associated with the development of colorectal cancer (CRC). However, the involvement of GM is CRC has mainly been demonstrated by metagenomic profiling studies showing the compositional difference between the GM of healthy individuals and that of CRC patients and not by directly studying isolated gut microbes. Thus, to discover novel gut microbes involved in CRC, we isolated the GM from the feces of healthy individuals and evaluated its anti-CRC activity in vitro and in vivo. After GM isolation, cell-free supernatants (CFSs) were prepared from the isolated gut microorganisms to efficiently screen a large amount of the GM for anti-proliferative ability in vitro. Our results showed that the CFSs of 21 GM isolates had anti-proliferative activity against human colon cancer HCT 116 cells. Of these 21 GM isolates, GM07 was chosen for additional study because it had the highest anti-cancer activity against mouse colon cancer CT 26 cells in vitro and was further evaluated in a CT 26 allograft mouse model in vivo. GM07 was identified as Odoribacter splanchnicus through phylogenetic analysis based on 16S rRNA gene sequencing. Further investigation determined that the CFS of O. splanchnicus (OsCFS) induced anti-proliferative activity via apoptosis, but not cell cycle arrest. Moreover, GC/MS analysis suggested that the putative active molecule in OsCFS is malic acid. Finally, in the CRC mouse model, peri-tumoral injection of OsCFS significantly decreased CRC formation, compared to the control group. Altogether, these findings will provide valuable information for the discovery of potential probiotic candidates that inhibit CRC.

Obesity is an important health concern in humans and dogs. It can cause a variety of secondary problems, including low bacterial diversity. Several approaches have been tried to solve this problem; one of them is probiotic supplementation. Lactobacillus gasseri BNR17 is derived from breast milk and has been proven to be effective for obesity in humans. However, there have been no studies using a synbiotic preparation containing L. gasseri BNR17 for obesity management in dogs. Therefore, the present study evaluated the effectiveness of a synbiotic preparation containing L. gasseri BNR17 in reducing body fat in obese dogs. A group of obese dogs were fed a synbiotic preparation for 10 weeks. Obesity variables included body weight, body condition score, subcutaneous fat thickness, subcutaneous fat mass and proportion of the fat mass. In addition, feces collected at 0-week and 10-week time points were analyzed for the intestinal microbiome. Results showed a significant decrease in body weight, body condition score, and subcutaneous fat mass and proportion at the level of the third lumbar vertebra. Diversity and functional analysis of the microbiota in obese dogs showed increased microbial diversity, and increased abundance of metabolism of carbohydrate, and lipid after supplementation with a synbiotic preparation. This study was conducted as a pilot study, and the results demonstrated that a synbiotic preparation containing L. gasseri BNR17 may play a role in reducing body fat and resolving the obesity in dogs.

Category: Trauma; Ankle Introduction/Purpose: Isolated lateral malleolus fracture is one of the most common fractures. However, there is controversy regarding the rehabilitation protocols used after surgery. In particular, the initiation point for the weight bearing has not been standardized. In the present study, we investigated the prognostic difference between the immediate and delayed weight bearing on isolated lateral malleolus fractures. Methods: Retrospectively, the medical records of matched 50 and 41patients in the immediate and delayed weight-bearing groups were reviewed. All patients were treated with open reduction and internal fixation using an anatomical locking compression plate. In the immediate weight-bearing group (IWB), tolerable weight bearing (i.e., that can be endured immediately after surgery with crutches) was permitted. In the delayed weight-bearing group (DWB), weight bearing was completely restricted for 4 weeks after surgery. Ankle motion exercise were permitted in both groups since postoperative day. Radiographic assessment data and the clinical outcomes were reviewed. Results: There were no significant differences noted in the radiographic assessments and complications between the two groups. Significant differences of shortening the return to work and hospital stays of IWB rehabilitation protocol were confirmed(5.7 vs. 8.0 days, 6.0 vs. 8.0 days in the IBW and DWB groups, respectively). In FAOS, there was a significant difference in the sport factor evaluated at postoperative 3 months(75.5 vs 68.5, respectively). Conclusion: There were no significant differences between the two groups in the postoperative radiological outcome and complications. And benefits of shortening the return to work and hospital stays of IWB rehabilitation protocol were confirmed. Conclusively, immediate weight bearing is recommended in patients with isolated lateral malleolus fracture, following anatomical reduction and firm fixation through surgery.

Category: Ankle; Sports; Trauma Introduction/Purpose: There are numerous studies recommending fibular groove deepening in the surgical treatment of peroneal tendon dislocation; however, there are some disadvantages to fibular groove deepening procedures. In this study, we evaluated the results of anatomic reattachment of the peroneal retinaculum without fibular groove deepening as a treatment for traumatic peroneal tendon dislocation. Methods: Thirty six patients with recurrent peroneal tendon dislocation, who underwent retinaculum repair without a fibular groove deepening procedure performed by two surgeons between March 2004 and March 2017, were enrolled in this study. Resubluxation of tendon, pain on inversion and eversion power of the ankle were monitored. The range of motion of inversion and eversion were measured and then compared to that of the contralateral side. AOFAS, VAS, FFI scores were obtained for all patients preoperatively and at the final follow-up. Postoperative complications such as infection, sural nerve injury, and recurrence were monitored. Results: Thirty four patients fully recovered without resubluxation of tendon. Two patients were injured again while playing soccer 6 months after the surgery and fast running 20 months after the surgery respectively. One patient had sural nerve injury. But the symptom was relieved at 6 months after the surgery. None of the patients had weakness of evertor. None of the patients had limited ankle motion. Mean AOFAS, VAS, FFI score improved significantly. Conclusion: In conclusion, for the treatment of recurrent dislocation of the peroneal tendon reattachment of the superior retinaculum only without groove deepening followed by proper rehabilitation is sufficient.

Category: Ankle, Ankle Arthritis Introduction/Purpose: The frequency of progression of osteoarthritis and persistence of symptoms in untreated osteochondral lesion of the talus (OCL) is not well known. We report the outcome of a nonoperative treatment for symptomatic OCL. Methods: This study included 142 patients with OCLs from 2003 to 2013. The patients did not undergo immobilization and had no restrictions of physical activities. The mean follow-up time was 6 (3–10) years. Initial MRI and CT confirmed OCL and showed lesion size, location, and stage of the lesion. Progression of osteoarthritis was evaluated by standing radiographs. In 83 patients, CT was performed at the final follow-up for analyses of the lesion size. We surveyed patients for limitations of sports activity, and Visual Analogue Scales (VAS), AOFAS, and SF-36 were assessed. Results: No patients had progression of osteoarthritis. The lesion size as determined by CT did not change in 69/83 patients, decreased in 5, and increased in 9. The mean VAS score of the 142 patients decreased from 3.8 to 0.9 (p < 0.001), the mean AOFAS ankle–hindfoot score increased from 86 to 93 (p < 0.001), and the mean SF-36 score increased from 52 to 72 (p < 0.001). Only 9 patients reported limitations of sports activity. The size and location of the lesion did not correlate with any of the outcome scores. Conclusion: Nonoperative treatment can be considered a good option for patients with OCL.

The Gram-positive rhizosphere bacterium Paenibacillus polymyxa promotes plant growth and produces various antibiotics. Herein, we review research on this species over the past two and a half decades, and focus on the mechanisms of P. polymyxa strain E681, isolated from barley roots in the South Korea in 1995. Strain E681 has outstanding growth-promoting effects on barley, cucumber, pepper, sesame, and Arabidopsis thaliana and produces antimicrobial compounds that protect plants against pathogenic fungi, oomycetes, and bacteria. Induced systemic resistance elicited by treating seeds or roots with strain E681 is a possible mechanism for protecting systemic plant tissues from biotic and other environmental stresses. Genome sequencing has broadened our horizons for antibiotic development and other industrial applications beyond agricultural use. At least six gene clusters for the biosynthesis of antibiotics have been discovered, including polymyxin (pmx), which was recently re-instated as an antibiotic of last resort against Gram-negative drug-resistant bacteria. Three groups of antibiotic synthetases include the gene clusters that encode one for the non-ribosomal peptide polymyxin, fusaricidin, and tridecaptin, another for the lantibiotic paenilan, and the third for a polyketide. We successfully introduced the pmx gene cluster into the surrogate host Bacillus subtilis and created polymyxin derivatives by domain swapping. Furthermore, various E681 derivatives, including a high fusaricidin producer and strains lacking multi-antibiotics production, have been constructed by random mutagenesis and genome engineering. Thus, E681 is an important bacterium that contributes to both plant and human health.

In Bacillus subtilis, large genomic deletions have been carried out for genome reduction, antibiotic overproduction, and heterologous protein overexpression. In view of the eco-friendliness of B. subtilis, it is critical that engineering preserves its food-grade status and avoids leaving foreign DNA in the genome. Existing methods of generating large genomic deletions leave antibiotic resistance markers or display low mutation efficiency. In this study, we introduced a clustered regularly interspaced short palindromic repeat-derived genome engineering technique to develop a highly efficient method of generating large genomic deletions in B. subtilis without any trace of foreign DNA. Using our system, we produced 38 kb plipastatin-synthesizing pps operon deletion with 80% efficiency. The significant increase in mutation efficiency was due to plasmids-delivered Streptococcus pyogenes-originated SpCas9, target-specific sgRNA and a donor DNA template, which produces SpCas9/sgRNA endonuclease complex continuously for attacking target chromosome until the mutagenic repair occurs. Our system produced single-gene deletion in spo0A (∼100%), point mutation (∼68%) and GFP gene insertion (∼97%) in sigE and demonstrated its broad applicability for various types of site-directed mutagenesis in B. subtilis.

In a previous study, we developed an E1 monobody specific for the tumor biomarker hEphA2 [PLoS ONE (2015) 10(7): e0132976]. E1 showed potential as a molecular probe for in vitro and in vivo targeting of cancers overexpressing hEphA2. In the present study, we constructed expression vectors for E1 conjugated to optical reporters such as Renilla luciferase variant 8 (Rluc8) or enhanced green fluorescent protein (EGFP) and purified such recombinant proteins by affinity chromatography in E. coli. E1-Rluc8 and E1-EGFP specifically bound to hEphA2 in human prostate cancer PC3 cells but not in human cervical cancer HeLa cells, which express hEphA2 at high and low levels, respectively. These recombinant proteins maintained >40% activity in mouse serum at 24 h. In vivo optical imaging for 24 h did not detect E1-EGFP signals, whereas E1-Rluc8 showed tumor-specific luminescence signals in PC3 but not in HeLa xenograft mice. E1-Rluc8 signals were detected at 4 h, peaked at 12 h, and were undetectable at 24 h. These results suggest the potential of E1-Rluc8 as an EphA2-specific optical imaging agent.

Monobodies are binding scaffold proteins originating from a human fibronectin domain III (Fn3) scaffold that can be easily engineered with specificity and affinity. Human EphA2 (hEphA2) is an early detection marker protein for various tumors including lung, breast, and colon cancer. In this study, we isolated two hEphA2-specific monobodies (E1 and E10) by screening a yeast surface display library. They showed the same amino acid sequence except in the DE loop and had high affinity (~2 nM Kd) against hEphA2. E1 bound only hEphA2 and mEphA2, although it bound hEphA2 with an affinity 2-fold higher than that of mEphA2. However, E10 also bound the mEphA6 and mEphA8 homologs as well as hEphA2 and mEphA2. Thus, E1 but not E10 was highly specific for hEphA2. E1 specifically bound human cells and xenograft tumor tissues expressing hEphA on the cell surface. In vivo optical imaging showed strong targeting of Cy5.5-labeled E1 to mouse tumor tissue induced by PC3 cells, a human prostate cancer cell line that expresses a high level of hEphA2. In conclusion, the highly specific monobody E1 is useful as a hEphA2 probe candidate for in vivo diagnosis and therapy.