Microbial Community Changes in Silkworms Suspected of Septicemia and Identification of Serratia sp.

Diseases that occur in silkworms include soft rot, hardening disease, digestive diseases, and sepsis. However, research on the causes of bacterial diseases occurring in silkworms and the resulting changes in the microbial community is lacking. Therefore, we examined the morphological characteristics of sepsis and changes in the microbial community between silkworms that exhibit a unique odor and healthy silkworms; thus, we established a relationship between disease-causing microorganisms and sepsis. After producing a 16S rRNA amplicon library for samples showing sepsis, we obtained information on the microbial community present in silkworms using next-generation sequencing. Compared to that in healthy silkworms, in silkworms with sepsis, the abundance of the Firmicutes phylum was significantly reduced, while that of Proteobacteria was increased. Serratia sp. was dominant in silkworms with sepsis. After bacterial isolation, identification, and reinfection through the oral cavity, we confirmed this organism as the disease-causing agent; its mortality rate was 1.8 times higher than that caused by Serratia marcescens. In summary, we identified a new causative bacterium of silkworm sepsis through microbial community analysis and confirmed that the microbial community balance was disrupted by the aberrant proliferation of certain bacteria.

Molecular diagnosis of 5 silkworm strains endemic to South Korea using single-nucleotide polymorphisms selected from whole-genome sequences.

The domesticated silkworm, Bombyx mori Linnaeus (Lepidoptera: Bombycidae), often poses a challenge in strain identification due to similarities in morphology and genetic background. In South Korea, around 40 silkworm strains are classified as premium, including 5 endemic tri-molting strains: Goryeosammyeon, Sammyeonhonghoeback, Hansammyeon, Sun7ho, and Sandongsammyeon. These strains have potential for breeding programs in response to emerging industry demands, necessitating a reliable strain identification method. In this study, we established a molecular diagnosis approach for these 5 strains. We selected 2-4 single-nucleotide polymorphisms (SNPs) for each strain from whole-genome sequences of 39 strains, encompassing 37 previously studied and 2 newly added. These SNPs were utilized to construct decision trees for each endemic strain identification. The SNPs can be used to distinguish each target strain from the 38 nontarget strains by the tetra-primer amplification refractory mutation system-polymerase chain reaction, with the exception of HMS which needs the addition of PCR-restriction fragment length polymorphism method at the final step. This decision tree-based method using genomic SNPs, coupled with the 2 typing methods, produced consistent and accurate results, providing 100% accuracy. Additionally, the significant number of remaining SNPs identified in this study could be valuable for future diagnosis of the other strains.

Customized Integrating-Sphere System for Absolute Color Measurement of Silk Cocoon with Corrugated Microstructure.

Silk fiber, recognized as a versatile bioresource, holds wide-ranging significance in agriculture and the textile industry. During the breeding of silkworms to yield new varieties, optical sensing techniques have been employed to distinguish the colors of silk cocoons, aiming to assess their improved suitability across diverse industries. Despite visual comparison retaining its primary role in differentiating colors among a range of silk fibers, the presence of uneven surface texture leads to color distortion and inconsistent color perception at varying viewing angles. As a result, these distorted and inconsistent visual assessments contribute to unnecessary fiber wastage within the textile industry. To solve these issues, we have devised an optical system employing an integrating sphere to deliver consistent and uniform illumination from all orientations. Utilizing a ColorChecker, we calibrated the RGB values of silk cocoon images taken within the integrating sphere setup. This process accurately extracts the authentic RGB values of the silk cocoons. Our study not only helps in unraveling the intricate color of silk cocoons but also presents a unique approach applicable to various specimens with uneven surface textures.

Structural Characteristics and Properties of Cocoon and Regenerated Silk Fibroin from Different Silkworm Strains.

Silk has attracted the attention of researchers as a biomedical and cosmetic material because of its good biocompatibility and cytocompatibility. Silk is produced from the cocoons of silkworms, which have various strains. In this study, silkworm cocoons and silk fibroins (SFs) were obtained from ten silkworm strains, and their structural characteristics and properties were examined. The morphological structure of the cocoons depended on the silkworm strains. The degumming ratio of silk ranged from 22.8% to 28% depending on the silkworm strains. The highest and lowest solution viscosities of SF were shown by 9671 and 9153, respectively, showing a 12-fold difference. The silkworm strains of 9671, KJ5, and I-NOVI showed a two-fold higher work of ruptures for the regenerated SF film than 181 and 2203, indicating that the silkworm strains considerably influence the mechanical properties of the regenerated SF film. Regardless of the silkworm strain, all silkworm cocoons showed good cell viability, making them suitable candidates for advanced functional biomaterials.

Visible light biophotosensors using biliverdin from Antheraea yamamai.

We report an endogenous photoelectric biomolecule and demonstrate that such a biomolecule can be used to detect visible light. We identify the green pigment abundantly present in natural silk cocoons of Antheraea yamamai (Japanese oak silkmoth) as biliverdin, using mass spectroscopy and optical spectroscopy. Biliverdin extracted from the green silk cocoons generates photocurrent upon light illumination with distinct colors. We further characterize the basic performance, responsiveness, and stability of the biliverdin-based biophotosensors at a photovoltaic device level using blue, green, orange, and red light illumination. Biliverdin could potentially serve as an optoelectric biomolecule toward the development of next-generation implantable photosensors and artificial photoreceptors.

Chitosan Extracted from the Biomass of Tenebrio molitor Larvae as a Sustainable Packaging Film.

Waste from non-degradable packaging materials poses a serious environmental risk and has led to interest in developing sustainable bio-based packaging materials. Sustainable packaging materials have been made from diverse naturally derived materials such as bamboo, sugarcane, and corn starch. In this study, we made a sustainable packaging film using chitosan extracted from the biomass of yellow mealworm (Tenebrio molitor) shell waste. The extracted chitosan was used to create films, cross-linked with citric acid (CA) and with the addition of glycerol to impart flexibility, using the solvent casting method. The successful cross-linking was evaluated using Fourier-Transform Infrared (FTIR) analysis. The CA cross-linked mealworm chitosan (CAMC) films exhibited improved water resistance with moisture content reduced from 19.9 to 14.5%. Improved barrier properties were also noted, with a 28.7% and 10.2% decrease in vapor permeability and vapor transmission rate, respectively. Bananas were selected for food preservation, and significant changes were observed over a duration of 10 days. Compared to the control sample, bananas packaged in CAMC pouches exhibited a lesser loss in weight because of excellent barrier properties against water vapor. Moreover, the quality and texture of bananas packaged in CAMC pouch remained intact over the duration of the experiment. This indicates that adding citric acid and glycerol to the chitosan structure holds promise for effective food wrapping and contributes to the enhancement of banana shelf life. Through this study, we concluded that chitosan film derived from mealworm biomass has potential as a valuable resource for sustainable packaging solutions, promoting the adoption of environmentally friendly practices in the food industry.

Antioxidant and cholesterol regulatory effect of flavonoid-rich silk sericin.

Cholesterol is a component of cell membranes and a precursor of hormones, and excess levels are associated with disease development; therefore, it must be maintained within the normal range. Silkworm cocoons are known to contain bioactive substances. Therefore, we compared the bioactivities of pigmented and white silkworm cocoons. Sericin extract of the Yeonnokjam (YN) variety, which contained a high flavonoid content, showed the highest antioxidant activity and inhibited cholesterol biosynthetic enzyme activity. YN-fed mice showed a 26% reduction in serum low-density lipoprotein cholesterol level. In addition, a 27% decrease in cholesterol accumulation in the liver was observed. Mechanistically, YN reduced the expression of 3-hydroxy-3-methylglutaryl-CoA reductase and acetyl-CoA acetyltransferase 2 proteins by 34 and 13%, respectively. In conclusion, YN suppresses cholesterol synthesis in the liver and stimulates bile acid secretion, which contributes to reduction in cholesterol levels, suggesting its potential as a cholesterol-lowering agent.

Modulatory Effects of the Kuwanon-Rich Fraction from Mulberry Root Bark on the Renin-Angiotensin System.

In this study, we investigated the anti-hypertensive properties of mulberry products by modulating the renin-angiotensin system (RAS). Comparative analysis showed that the ethyl acetate fractions, particularly from the Cheongil and Daeshim cultivars, contained the highest levels of polyphenols and flavonoids, with concentrations reaching 110 mg gallic acid equivalent (GE)/g and 471 mg catechin equivalent (CE)/g of extract, respectively. The ethyl acetate fraction showed superior angiotensin-converting enzyme (ACE) inhibitory activity, mainly because of the presence of the prenylated flavonoids kuwanon G and H. UPLC/Q-TOF-MS analysis identified kuwanon G and H as the primary active components, which significantly contributed to the pharmacological efficacy of the extract. In vivo testing of mice fed a high-salt diet showed that the ethyl acetate fraction substantially reduced the heart weight and lowered the serum renin and angiotensinogen levels by 34% and 25%, respectively, highlighting its potential to modulate the RAS. These results suggested that the ethyl acetate fraction of mulberry root bark is a promising candidate for the development of natural ACE inhibitors. This finding has significant implications for the management of hypertension through RAS regulation and the promotion of cardiovascular health in the functional food industry.

Use of a Silkworm (Bombyx mori) Larvae By-Product for the Treatment of Atopic Dermatitis: Inhibition of NF-κB Nuclear Translocation and MAPK Signaling.

Atopic dermatitis (AD) is a long-lasting inflammatory skin disease that contributes to the global health burden and impacts 10-20% of the world's population. In this study, we determined the anti-AD effect of a by-product of silkworm (Bombyx mori) larval powder, strain Yeonnokjam (SLPY), as a sustainable, natural source for the development of therapeutic agents for AD. HaCaT cells were used to assess the in vitro anti-inflammatory activity of SLPY, and a 1-chloro-2,4-dinitrobenzene (DNCB)-induced mouse model was used to study the in vivo anti-AD effects. SLPY treatment downregulated the expression of the inflammatory cytokines TNF-α, IL1ß, IL-8, and Cox-2 in stimulated HaCaT cells. Similarly, the topical application of SLPY in DNCB-treated mice downregulated the expression of inflammatory cytokines and proteins while ameliorating the clinical features of AD. Further, SLPY treatment inhibited the nuclear translocation of NF-κb p65, thereby supporting the efficacy of SLPY in the treatment of AD.

Whole-genome sequences of 37 breeding line Bombyx mori strains and their phenotypes established since 1960s.

Bombyx mori is a key insect in the sericulture industry and one of the very important economic animals that are responsible for not only the livelihood of many farmers internationally but also expended biomedical use. The National Institute of Agricultural Sciences of the Rural Development Administration of Korea (NIAS, RDA, Korea) has been collecting silkworm resources with various phenotypic traits from the 1960s and established breeding lines for using them as genetic resources. And these breeding line strains have been used to develop suitable F1 hybrid strains for specific use. In this study, we report the whole-genome sequences of 37 breeding line B. mori strains established over the past 60 years, along with the description of their phenotypic characteristics with photos of developmental stages. In addition, we report the example phenotypic characteristics of the F1-hybrid strain using these breeding line strains. We hope this data will be used as valuable resources to the related research community for studying B. mori and similar other insects.

Phylogeographic Relationships among Bombyx mandarina (Lepidoptera: Bombycidae) Populations and Their Relationships to B. mori Inferred from Mitochondrial Genomes.

We report 37 mitochondrial genome (mitogenome) sequences of Bombyx mori strains (Lepidoptera: Bombycidae) and four of B. mandarina individuals, each preserved and collected, respectively, in South Korea. These mitogenome sequences combined with 45 public data showed a substantial genetic reduction in B. mori strains compared to the presumed ancestor B. mandarina, with the highest diversity detected in the Chinese origin B. mori. Chinese B. mandarina were divided into northern and southern groups, concordant to the Qinling-Huaihe line, and the northern group was placed as an immediate progenitor of monophyletic B. mori strains in phylogenetic analyses, as has previously been detected. However, one individual that was in close proximity to the south Qinling-Huaihe line was exceptional, belonging to the northern group. The enigmatic South Korean population of B. mandarina, which has often been regarded as a closer genetic group to Japan, was most similar to the northern Chinese group, evidencing substantial gene flow between the two regions. Although a substantial genetic divergence is present between B. mandarina in southern China and Japan, a highly supported sister relationship between the two regional populations may suggest the potential origin of Japanese B. mandarina from southern China instead of the Korean peninsula.

Edible Matrix Code with Photogenic Silk Proteins.

Counterfeit medicines are a healthcare security problem, posing not only a direct threat to patient safety and public health but also causing heavy economic losses. Current anticounterfeiting methods are limited due to the toxicity of the constituent materials and the focus of secondary packaging level protections. We introduce an edible, imperceptible, and scalable matrix code of information representation and data storage for pharmaceutical products. This matrix code is digestible as it is composed of silk fibroin genetically encoded with fluorescent proteins produced by ecofriendly, sustainable silkworm farming. Three distinct fluorescence emission colors are incorporated into a multidimensional parameter space with a variable encoding capacity in a format of matrix arrays. This code is smartphone-readable to extract a digitized security key augmented by a deep neural network for overcoming fabrication imperfections and a cryptographic hash function for enhanced security. The biocompatibility, photostability, thermal stability, long-term reliability, and low bit error ratio of the code support the immediate feasibility for dosage-level anticounterfeit measures and authentication features. The edible code affixed to each medicine can serve as serialization, track and trace, and authentication at the dosage level, empowering every patient to play a role in combating illicit pharmaceuticals.

Biocompatible fluorescent silk fibroin bioink for digital light processing 3D printing.

Chemically modified silk fibroin (SF) bioink has been used for three-dimensional (3D) bioprinting in tissue engineering because of its biocompatibility and printability. Also, fluorescent silk fibroin (FSF) from transgenic silkworms has been recently applied in biomedicine because of its fluorescence property. However, the fabrication of fluorescent hydrogel from FSF has not been elucidated. In this study, we showed the fabrication of a digital light processing (DLP) printable bioink from a chemically modified FSF. This bioink was fabricated by covalent conjugation of FSF and glycidyl methacrylate (GMA) and can be printed into various structures, such as the brain, ear, hand, lung, and internal organs. The physical properties of glycidyl methacrylated fluorescent silk fibroin (FSGMA) hydrogel was like the glycidyl methacrylated non-fluorescent silk fibroin (SGMA) hydrogel. The FSGMA hydrogel significantly retains its fluorescence property and has excellent biocompatibility. All these properties make FSGMA hydrogel a potent tool in encapsulated cell tracking and observing the scaffolds' degradation in vivo. This study suggested that our 3D DLP printable FSF bioink could play a promising role in the biomedical field.

Anterior cruciate ligament reconstruction using 4-strand hamstring autograft: conventional single-bundle technique versus oval-footprint technique.

PURPOSE: The purpose of this study was to compare short-term results of conventional anterior cruciate ligament (ACL) reconstruction with oval-footprint (modified) single-tunnel ACL reconstruction with 4-strand hamstring autograft. METHODS: A prospective comparative study was performed in 74 consecutive subjects who underwent ACL reconstruction with the conventional technique (group I, 40 cases) or the modified technique (group II, 34 cases), in which the entrances of the femoral and tibial tunnels were more elongated, by use of 4-strand hamstring tendon. The Lachman test, pivot-shift test, range of motion, International Knee Documentation Committee classification, Lysholm score, and side-to-side differences were evaluated preoperatively and at the last follow-up. The Tegner activity scale was evaluated before injury and at the last follow-up. RESULTS: There were 38 patients in group I and 32 in group II who were followed up for at least 2 years (mean follow-up, 32.4 months). At the last follow-up, Lachman test results were negative in 34 in group I and 30 in group II (P = .624) and the pivot-shift test was negative in 32 in group I and 30 in group II (P = .397). Mean range of motion of the injured knee was 142.2° in group I and 141.9° in group II (P = .771). The International Knee Documentation Committee classification was A or B in 37 in group I and 31 in group II (P = .872). The median Lysholm score was 94 in group I and 96 in group II (P = .048). The mean side-to-side difference averaged 2.08 mm in group I and 2.07 mm in group II (P = .943). The median score on the Tegner activity scale was 6 in group I and 6 in group II (P = .968). CONCLUSIONS: The Lysholm score in the modified-technique group at the last follow-up was better than that in the conventional-technique group in terms of statistical significance, but this may not be clinically significant. LEVEL OF EVIDENCE: Level II, prospective comparative study.

Comparison between hamstring autograft and free tendon Achilles allograft: minimum 2-year follow-up after anterior cruciate ligament reconstruction using EndoButton and Intrafix.

PURPOSE: This study is to compare the clinical and radiographic results of anterior cruciate ligament (ACL) reconstruction with four-stranded autogenous hamstring tendon and two-stranded free tendon Achilles allograft fixed with EndoButton in the femoral tunnel and Intrafix in the tibial tunnel. MATERIALS AND METHODS: 106 patients diagnosed with ACL rupture underwent ACL reconstruction. Autogenous hamstring tendon was used in 33 patients (group I) and free tendon Achilles allografts were used in 32 patients (group II). Median age was 23 years old (20-51) in group I and 22 years old (20-55) in group II. Range of motion, Lachman test, Pivot shift test, IKDC score, Lysholm score and side-to-side difference (SSD) were evaluated preoperatively and at the last follow-up. Tegner activity scale was evaluated before injury and at the last follow-up. RESULTS: The mean follow-up periods were 28.1 months in group I and 31.6 months in group II. Range of motion of the knee was not different from that of the unaffected side in most cases except one flexion deficit in group I and three in group II (n.s.). One in group I and three in group II showed grade two or three laxity on Lachman test at the last follow-up. One in group I and three in group II showed clear positive results on Pivot shift test at the last follow-up. Thirty in group I and 26 in group II were classified to IKDC A or B at the last follow-up (n.s.). Median Lysholm scores were 98 (85-100) in group I and 99 (85-100) in group II at the last follow-up (n.s.). Median Tegner activity scales were 6 (5-9) in group I and 6 (4-9) in group II at the last follow-up (n.s.). The mean SSD at the last follow-up were 1.4 ± 2.0 mm in group I and 1.9 ± 2.4 mm in group II (n.s.). CONCLUSION: Clinical and radiological outcomes of ACL reconstruction with two-stranded free tendon Achilles allograft were comparable to those of four-stranded autogenous hamstring tendon. This technique is reasonable to accomplish good results without some weaknesses when using allograft with bone block. LEVEL OF EVIDENCE: Therapeutic randomized controlled prospective study, Level I.

Complete mitochondrial genome of the highly fecund Bombyx mori linnaeus, 1758 (Lepidoptera: Bombycidae) strain Jam 146.

To meet the increasing demands of the society in the current era, new strains of the domesticated silkworm Bombyx mori Linnaeus, 1758 (Lepidoptera: Bombycidae) are being continuously bred. Consequently, cataloging the genetic information of pure lines is essential. The strain Jam 146, whose larvae have atypical pale, crescent-shaped body markings, is an important breeding resource due to its excellent fecundity. In this study, we sequenced the mitochondrial genome (mitogenome) of this strain using next-generation sequencing. The complete genome of this strain has a gene arrangement typical of Lepidoptera. The length of the Jam 146 mitogenome (15,661 bp) is well within the range reported in other B. mori strains, i.e. between 15,629 (Baiyun strain, China) and 15,676 bp (Hukpyobeom strain, South Korea). However, the total length of protein-coding genes, 3,733 codons in Jam 146 and two other silkworm strains previously reported from South Korea, is 13 codons longer than that in other B. mori strains. Phylogenetic analysis of 22 silkworm strains from nine countries showed that the Jam 146 strain forms a strong cluster with three other strains from China, Japan, and South Korea, suggesting that after their split from a common ancestor, the evolutionary divergence among the silkworm strains in these countries has been limited.

Edible unclonable functions.

Counterfeit medicines are a fundamental security problem. Counterfeiting medication poses a tremendous threat to patient safety, public health, and the economy in developed and less developed countries. Current solutions are often vulnerable due to the limited security levels. We propose that the highest protection against counterfeit medicines would be a combination of a physically unclonable function (PUF) with on-dose authentication. A PUF can provide a digital fingerprint with multiple pairs of input challenges and output responses. On-dose authentication can verify every individual pill without removing the identification tag. Here, we report on-dose PUFs that can be directly attached onto the surface of medicines, be swallowed, and digested. Fluorescent proteins and silk proteins serve as edible photonic biomaterials and the photoluminescent properties provide parametric support of challenge-response pairs. Such edible cryptographic primitives can play an important role in pharmaceutical anti-counterfeiting and other security applications requiring immediate destruction or vanishing features.

Bombyx mori kynurenine 3-monooxygenase gene editing and insect molecular breeding using the clustered regularly interspaced short palindromic repeat/CRISPR associated protein 9 system.

Genome editing by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated protein (Cas)9, a third-generation gene scissors, and molecular breeding at the genome level are attracting considerable attention as future breeding techniques. In the present study, genetic and phenotypic analyses were conducted to examine the molecular breeding of Bombyx mori through CRISPR/Cas9-mediated editing of the kynurenine 3-monooxygenase (KMO) gene. The synthesized guide RNAs (gRNAs) were analyzed using T7 endonuclease I after introduction into the BM-N silkworm cell line. To edit the silkworm gene, K1P gRNA, and Cas9 complexes were microinjected into silkworm embryos. After microinjection, the hatching rate and the incidence of mutation were determined as 18.1% and 60%, respectively. Gene mutation was verified in the heterozygous G0 generation, but no phenotypic change was observed; however, certain embryos and moths produced through sib-mating had significant differences compared to the wild-type. In successive generations, a distinct phenotypic change was also observed by continuous mating. Thus, although there are limitations in the phenotypic expression in breeding through the induction of deletion mutations, as in the present study, the process is believed to yield successful results within a shorter period compared to traditional breeding and is safer than transgenic technology.

Photoelectric Silk via Genetic Encoding and Bioassisted Plasmonics.

Genetically encoded photoelectric silk that can convert photons to electrons (light to electricity) over a wide visible range in a self-power mode is reported. As silk is a versatile host material with electrical conductivity, biocompatibility, and processability, a photoelectric protein is genetically fused with silk by silkworm transgenesis. Specifically, mKate2, which is conventionally known as a far-red fluorescent protein, is used as a photoelectric protein. Characterization of the electrochemical and optical properties of mKate2 silk allows designing a photoelectric measurement system. A series of in situ photocurrent experiments support the sensitive and stable performance of photoelectric conversion. In addition, as a plasmonic nanomaterial with a broad spectral resonance, titanium nitride (TiN) nanoparticles are biologically hybridized into the silk glands, taking full advantage of the silkworms' open circulatory system as well as the absorption band of mKate2 silk. This biological hybridization via direct feeding of TiN nanoparticles further enhances the overall photoelectric conversion ability of mKate2 silk. It is envisioned that the biologically derived photoelectric protein, its ecofriendly scalable production by transgenic silkworms, and the bioassisted plasmonic hybridization can potentially broaden the biomaterial choices for developing next-generation biosensing, retina prosthesis, and neurostimulation applications.

Complete mitochondrial genome of the silkworm strain, Chilseongjam Bombyx mori (Lepidoptera: Bombycidae), with a unique larval body marking.

Recently, a new silkworm strain with a peculiar larval marking and rare cocoon colour was bred in Korea for educational learning and exhibition. In order to obtain the genetic information of the newly bred strain, Chilseongjam Bombyx mori (Lepidoptera: Bombycidae), its complete mitochondrial genome (mitogenome) was sequenced. The mitogenome is 15,660 bp in length, contains a typical set of genes, and has gene arrangement and composition typical of Lepidoptera. However, the Chilseongjam strain mitogenome is 4-36 bp longer than 19 other strains originating from other countries and 16 bp shorter than the whole genome of a Korean Hukpyobeom strain. In particular, the Chilseongjam strain has an intergenic spacer sequence that is shorter than that of the Hukpyobeom strain at the tRNAHis and ND4 junction as it has fewer microsatellite-like AT repeats. Phylogenetic analyses conducted using a total of 21 silkworm strains originating from nine countries revealed a few subgroups with moderate-to-high nodal support (80-94%). The Korean Chilseongjam strain formed a relatively strong subgroup (85%) with a Japanese strain (J106) instead of the Korean Hukpyobeom strain.