Attempting to Create a Pathway to 15-Deacetylcalonectrin with Limited Accumulation in Cultures of Fusarium Tri3 Mutants: Insight into Trichothecene Biosynthesis Machinery.

The compound 15-deacetylcalonectrin (15-deCAL) is a common pathway intermediate in the biosynthesis of Fusarium trichothecenes. This tricyclic intermediate is metabolized to calonectrin (CAL) by trichothecene 15-O-acetyltransferase encoded by Tri3. Unlike other trichothecene pathway Tri gene mutants, the Δtri3 mutant produces lower amounts of the knocked-out enzyme's substrate 15-deCAL, and instead, accumulates higher quantities of earlier bicyclic intermediate and shunt metabolites. Furthermore, evolutionary studies suggest that Tri3 may play a role in shaping the chemotypes of trichothecene-producing Fusarium strains. To better understand the functional role of Tri3p in biosynthesis and evolution, we aimed to develop a method to produce 15-deCAL by using transgenic Fusarium graminearum strains derived from a trichothecene overproducer. Unfortunately, introducing mutant Tri3, encoding a catalytically impaired but structurally intact acetylase, did not improve the low 15-deCAL production level of the ΔFgtri3 deletion strain, and the bicyclic products continued to accumulate as the major metabolites of the active-site mutant. These findings are discussed in light of the enzyme responsible for 15-deCAL production in trichothecene biosynthesis machinery. To efficiently produce 15-deCAL, we tested an alternative strategy of using a CAL-overproducing transformant. By feeding a crude CAL extract to a Fusarium commune strain that was isolated in this study and capable of specifically deacetylating C-15 acetyl, 15-deCAL was efficiently recovered. The substrate produced in this manner can be used for kinetic investigations of this enzyme and its possible role in chemotype diversification.

A Role in 15-Deacetylcalonectrin Acetylation in the Non-Enzymatic Cyclization of an Earlier Bicyclic Intermediate in Fusarium Trichothecene Biosynthesis.

The trichothecene biosynthesis in Fusarium begins with the cyclization of farnesyl pyrophosphate to trichodiene, followed by subsequent oxygenation to isotrichotriol. This initial bicyclic intermediate is further cyclized to isotrichodermol (ITDmol), a tricyclic precursor with a toxic trichothecene skeleton. Although the first cyclization and subsequent oxygenation are catalyzed by enzymes encoded by Tri5 and Tri4, the second cyclization occurs non-enzymatically. Following ITDmol formation, the enzymes encoded by Tri101, Tri11, Tri3, and Tri1 catalyze 3-O-acetylation, 15-hydroxylation, 15-O-acetylation, and A-ring oxygenation, respectively. In this study, we extensively analyzed the metabolites of the corresponding pathway-blocked mutants of Fusarium graminearum. The disruption of these Tri genes, except Tri3, led to the accumulation of tricyclic trichothecenes as the main products: ITDmol due to Tri101 disruption; a mixture of isotrichodermin (ITD), 7-hydroxyisotrichodermin (7-HIT), and 8-hydroxyisotrichodermin (8-HIT) due to Tri11 disruption; and a mixture of calonectrin and 3-deacetylcalonectrin due to Tri1 disruption. However, the ΔFgtri3 mutant accumulated substantial amounts of bicyclic metabolites, isotrichotriol and trichotriol, in addition to tricyclic 15-deacetylcalonectrin (15-deCAL). The ΔFgtri5ΔFgtri3 double gene disruptant transformed ITD into 7-HIT, 8-HIT, and 15-deCAL. The deletion of FgTri3 and overexpression of Tri6 and Tri10 trichothecene regulatory genes did not result in the accumulation of 15-deCAL in the transgenic strain. Thus, the absence of Tri3p and/or the presence of a small amount of 15-deCAL adversely affected the non-enzymatic second cyclization and C-15 hydroxylation steps.

Mechanism of Secondary Glaucoma Development in HTLV-1 Uveitis.

Human T-cell lymphotropic virus type 1 (HTLV-1) was the first retrovirus identified as the causative agent of human diseases, such as adult T-cell leukemia, HTLV-1-associated myelopathy, and HTLV-1 uveitis (HU). HU is one of the most frequent ocular inflammatory diseases in endemic areas, which has raised considerable public health concerns. Approximately 30% of HU patients develop secondary glaucoma, which is higher than the general uveitis incidence. We therefore investigated the mechanism underlying the high incidence of glaucoma secondary to HU in vitro. After contact with HTLV-1-producing T cells (MT-2), human trabecular meshwork cells (HTMCs) were infected. The infected cells increased in number, and nuclear factor (NF)-κB expression was activated. Contact between MT-2 cells and HTMCs resulted in significantly upregulated production of inflammatory cytokines, such as IL-6, and chemokines, such as CXCL10, CCL2, and CXCL-8. These findings indicate that the mechanism underlying secondary glaucoma in HU may involve proliferation of trabecular meshwork tissue after contact with HTLV-1-infected cells, resulting in decreased aqueous humor outflow. Upregulated production of inflammatory cytokines and chemokines simultaneously disrupts the normal trabecular meshwork function. This mechanism presumably leads to increased intraocular pressure, eventually resulting in secondary glaucoma.

4-O-Glucosylation of Trichothecenes by Fusarium Species: A Phase II Xenobiotic Metabolism for t-Type Trichothecene Producers.

The t-type trichothecene producers Fusarium sporotrichioides and Fusarium graminearum protect themselves against their own mycotoxins by acetylating the C-3 hydroxy group with Tri101p acetylase. To understand the mechanism by which they deal with exogenously added d-type trichothecenes, the Δtri5 mutants expressing all but the first trichothecene pathway enzymes were fed with trichodermol (TDmol), trichothecolone (TCC), 8-deoxytrichothecin, and trichothecin. LC-MS/MS and NMR analyses showed that these C-3 unoxygenated trichothecenes were conjugated with glucose at C-4 by α-glucosidic linkage. As t-type trichothecenes are readily incorporated into the biosynthetic pathway following the C-3 acetylation, the mycotoxins were fed to the ΔFgtri5ΔFgtri101 mutant to examine their fate. LC-MS/MS and NMR analyses demonstrated that the mutant conjugated glucose at C-4 of HT-2 toxin (HT-2) by α-glucosidic linkage, while the ΔFgtri5 mutant metabolized HT-2 to 3-acetyl HT-2 toxin and T-2 toxin. The 4-O-glucosylation of exogenously added t-type trichothecenes appears to be a general response of the ΔFgtri5ΔFgtri101 mutant, as nivalenol and its acetylated derivatives appeared to be conjugated with hexose to some extent. The toxicities of 4-O-glucosides of TDmol, TCC, and HT-2 were much weaker than their corresponding aglycons, suggesting that 4-O-glucosylation serves as a phase II xenobiotic metabolism for t-type trichothecene producers.

Accumulation of 4-Deoxy-7-hydroxytrichothecenes, but Not 4,7-Dihydroxytrichothecenes, in Axenic Culture of a Transgenic Nivalenol Chemotype Expressing the NX-Type FgTri1 Gene.

Fusarium graminearum species complex produces type B trichothecenes oxygenated at C-7. In axenic liquid culture, F. graminearum mainly accumulates one of the three types of trichothecenes, namely 3-acetyldeoxyinvalenol, 15-acetyldeoxyinvalenol, or mixtures of 4,15-diacetylnivalenol/4-acetylnivalenol, depending on each strain's genetic background. The acetyl groups of these trichothecenes are slowly deacetylated to give deoxynivalenol (DON) or nivalenol (NIV) on solid medium culture. Due to the evolution of F. graminearum FgTri1, encoding a cytochrome P450 monooxygenase responsible for hydroxylation at both C-7 and C-8, new derivatives of DON, designated as NX-type trichothecenes, have recently emerged. To assess the risks of emergence of new NX-type trichothecenes, we examined the effects of replacing FgTri1 in the three chemotypes with FgTri1_NX chemotype, which encodes a cytochrome P450 monooxygenase that can only hydroxylate C-7 of trichothecenes. Similar to the transgenic DON chemotypes, the transgenic NIV chemotype strain accumulated NX-type 4-deoxytrichothecenes in axenic liquid culture. C-4 oxygenated trichothecenes were marginal, despite the presence of a functional FgTri13 encoding a C-4 hydroxylase. At present, outcrossing of the currently occurring NX chemotype with NIV chemotype strains of F. graminearum in the natural environment likely will not yield a new strain that produces a C-4 oxygenated NX-type trichothecene.

Recent Progress in the Development of Disinfectants from Scallop Shell-Derived Calcium Oxide for Clinical and Daily Use.

The current pandemic of novel coronavirus disease (COVID-19) has highlighted the importance of disinfectants. As a raw material for next-generation disinfectants, scallop shell-derived calcium oxide (CaO) has been revealed to exhibit significant virucidal and microbicidal activities and is compatible with living tissues and the environment. This minireview summarizes recent progress in the development of disinfectants from scallop shell-CaO, focusing especially on studies of clinical and daily use applications. We describe the preparation, basic characteristics, and virucidal and microbicidal activities of scallop shell-CaO disinfectants. Furthermore, their applications in the disinfection of contaminated masks and the treatment of infected wounds are briefly introduced.

Efficacy of Bioshell Calcium Oxide Water as Disinfectants to Enable Face Mask Reuse.

Bioshell calcium oxide (BiSCaO) is derived from scallop shells and after heat treatment exhibits broad microbicidal activity. BiSCaO Water is a disinfectant prepared by collecting the aqueous layer after adding BiSCaO powder to water, is colorless and transparent, and has a pH of 12.8. We compared the utility of commercially available BiSCaO Water, ethanol, sodium hypochlorite, hypochlorous acid and hydrogen peroxide solutions as sterilization agents to enable the reuse of surgical and N95 face masks. The microbicidal efficacy of each disinfectant was evaluated using pieces of surgical and N95 face masks contaminated with normal bacterial flora. The results suggest that BiSCaO Water has excellent disinfection activity toward contaminated polypropylene masks and has minimal adverse effect on the structure of non-woven masks.

Safety of Concentrated Bioshell Calcium Oxide Water Application for Surface and Skin Disinfections against Pathogenic Microbes.

Immediately post-production, commercially available bioshell calcium oxide (BiSCaO) water is colorless, transparent, and strongly alkaline (pH 12.8), and is known to possess deodorizing properties and broad microbicidal activity. However, BiSCaO Water may represent a serious safety risk to the living body, given the strong alkalinity. This study aimed to investigate the safety of BiSCaO Water for use as an antiseptic/disinfectant despite concerns regarding its high alkalinity. The change over time in pH of BiSCaO Water was measured during air contact (stirring BiSCaO Water in ambient air). When sprayed on metal, plastic, wood piece, paper, and skin surfaces, the pH of BiSCaO Water decreased rapidly, providing a white powder coating upon drying. Scanning electron microscopy images, energy dispersive X-ray elemental mapping, and X-ray diffractograms showed that the dried powder residues of BiSCaO Water were composed primarily of calcium carbonate. These results suggested that BiSCaO Water is a potent reagent that may overcome the obstacles of being strongly alkaline, making this material appropriate for use in disinfection against pathogenic microbes.

Haloarcula mannanilytica sp. nov., a galactomannan-degrading haloarchaeon isolated from commercial salt.

A mannan-degrading halophilic archaeal strain, MD130-1T, was isolated from a commercial salt sample. Cells were motile, rod-shaped, and stained Gram-negative. Colonies were pink pigmented. Strain MD130-1T was able to grow at 1.5-4.6 M NaCl (optimum, 3.6 M) at pH 6.0-8.0 (optimum, pH 7.0) and at 25-50 °C (optimum, 40 °C). The DNA G+C content was 62.1 mol% (genome). The orthologous 16S rRNA gene sequence showed the highest similarity (99.4 %) to those of Haloarcula japonica JCM 7785T and Haloarcula hispanica JCM 8911T. The values of genome relatedness between strain MD130-1T and Haloarcula species were 84.33-85.96 % in ANIb and 30.4-32.9 % using GGDC formula 2. The polar lipids of strain MD130-1T were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and triglycosyl diether-2. Based on the results of phenotypic and phylogenetic analyses, the strain represents a new species of the genus Haloarcula, for which the name Haloarcula mannanilytica sp. nov. is proposed. The type strain is MD130-1T (=JCM 33835T=KCTC 4287T) isolated from commercial salt made in Ishikawa prefecture, Japan.

Development of Novel Heparin/Protamine Nanoparticles Useful for Delivery of Exogenous Proteins In Vitro and In Vivo.

We previously reported that heparin/protamine particles (LHPPs) produced as nanoparticles through simple mixing of raw materials exhibit sustained protein release and can be retained in cells. In the present study, we modified LHPPs without employing any organic synthetic approach. The resulting LHPPs were re-named as improved LHPPs (i-LHPPs) and have the ability to retain cell-penetrating peptides (GRKKRRQRRRPPQ) based on electrostatic interactions. We examined whether i-LHPPs can introduce exogenous proteins (i.e., lacZ protein encoding bacterial ß-galactosidase) into cultured cells in vitro, or into murine hepatocytes in vivo through intravenous injection to anesthetized mice. We found an accumulation of the transferred protein in both in vitro cultured cells and in vivo hepatocytes. To the best of our knowledge, reports of successful in vivo delivery to hepatocytes are rare. The i-LHPP-based protein delivery technique will be useful for in vivo functional genetic modification of mouse hepatocytes using Cas9 protein-mediated genome editing targeting specific genes, leading to the creation of hepatic disease animal models for research that aims to treat liver diseases.

Hydrodynamics-Based Transplacental Delivery as a Useful Noninvasive Tool for Manipulating Fetal Genome.

We previously demonstrated that the injection of pregnant wild-type female mice (carrying enhanced green fluorescent protein (EGFP)-expressing transgenic fetuses) at embryonic day (E) 12.5 with an all-in-one plasmid conferring the expression of both Cas9 and guide RNA (targeted to the EGFP cDNA) complexed with the gene delivery reagent, resulted in some fetuses exhibiting reduced fluorescence in their hearts and gene insertion/deletion (indel) mutations. In this study, we examined whether the endogenous myosin heavy-chain α (MHCα) gene can be successfully genome-edited by this method in the absence of a gene delivery reagent with potential fetal toxicity. For this, we employed a hydrodynamics-based gene delivery (HGD) system with the aim of ensuring fetal gene delivery rates and biosafety. We also investigated which embryonic stages are suitable for the induction of genome editing in fetuses. Of the three pregnant females injected at E9.5, one had mutated fetuses: all examined fetuses carried exogenous plasmid DNA, and four of 10 (40%) exhibited mosaic indel mutations in MHCα. Gene delivery to fetuses at E12.5 and E15.5 did not cause mutations. Thus, the HGD-based transplacental delivery of a genome editing vector may be able to manipulate the fetal genomes of E9.5 fetuses.

Concentrated Bioshell Calcium Oxide (BiSCaO) Water Kills Pathogenic Microbes: Characterization and Activity.

Bioshell calcium oxide (BiSCaO) exhibits deodorizing properties and broad microbicidal activity. In this study, we examined possible utility of BiSCaO Water for that purpose. BiSCaO Water was prepared by adding 10 wt% BiSCaO to clean water and gently collecting the supernatant in a bottle. The same volume of clean water was gently poured onto the BiSCaO precipitate and the supernatant was gently collected in a bottle; this process was repeated fifty times. The produced BiSCaO Water contained nanoparticles (about 400-800 nm) composed of smaller nanoparticles (100-200 nm), and was colorless and transparent, with a pH > 12.7. In vitro assays demonstrated that BiSCaO Water eliminated more than 99.9% of influenza A (H1N1) and Feline calicivirus, Escherichia coli such as NBRC 3972 and O-157:H7, Pseudomonas aeruginosa, Salmonella, and Staphylococcus aureus within 15 min. We compared BiSCaO Water with the other microbicidal reagents such as ethanol, BiSCaO, BiSCa(OH)2 suspensions, povidone iodine, NaClO, BiSCaO dispersion and colloidal dispersion with respect to deodorization activity and microbicidal efficacy. The results showed that BiSCaO Water was a potent reagent with excellent deodorization and disinfection activities against pathogenic bacteria and viruses (including both enveloped and nonenveloped viruses).

Ultraviolet Irradiation Enhances the Microbicidal Activity of Silver Nanoparticles by Hydroxyl Radicals.

It is known that silver has microbicidal qualities; even at a low concentration, silver is active against many kinds of bacteria. Silver nanoparticles (AgNPs) have been extensively studied for a wide range of applications. Alternately, the toxicity of silver to human cells is considerably lower than that to bacteria. Recent studies have shown that AgNPs also have antiviral activity. We found that large amounts of hydroxyl radicals-highly reactive molecular species-are generated when AgNPs are irradiated with ultraviolet (UV) radiation with a wavelength of 365 nm, classified as ultraviolet A (UVA). In this study, we used electron spin resonance direct detection to confirm that UV irradiation of AgNPs produced rapid generation of hydroxyl radicals. As hydroxyl radicals are known to degrade bacteria, viruses, and some chemicals, the enhancement of the microbicidal activity of AgNPs by UV radiation could be valuable for the protection of healthcare workers and the prevention of the spread of infectious diseases.

Biosynthetic gene cluster identification and biological activity of lucilactaene from Fusarium sp. RK97-94.

We identified the biosynthetic gene cluster for lucilactaene, a cell cycle inhibitor from a filamentous fungus Fusarium sp. RK 97-94. The luc1 knockout strain accumulated demethylated analogs, indicating the involvement of Luc1 methyltransferase in lucilactaene biosynthesis. Lucilactaene showed potent antimalarial activity. Our data suggested that methylation and ether ring formation are essential for its potent antimalarial activity.

Substrate specificities of Fusarium biosynthetic enzymes explain the genetic basis of a mixed chemotype producing both deoxynivalenol and nivalenol-type trichothecenes.

Fusarium species are traditionally grouped into type A and type B trichothecene producers based on structural differences in the mycotoxin they synthesize. The type B trichothecene-producing Fusarium graminearum strains are further divided into 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), and nivalenol (NIV) chemotypes. The former two chemotypes, collectively termed a deoxynivalenol (DON) chemotype, evolved from a NIV chemotype by inactivation of FgTri13, which encodes trichothecene C-4 hydroxylase, during the evolutionary process. Despite stable overexpression of FgTri13, however, both 3-acetylnivalenol (3-ANIV) and 3-ADON accumulate equally in shake flask culture of a transgenic 3-ADON chemotype. In this study, we investigated why the "3-ANIV chemotype" could not be obtained using this strategy. When analysis was extended to the transgenic NIV chemotype, in which FgTri7 C-4 acetylase gene was disrupted and FgTri8 deacetylase gene was replaced with the 3-ADON chemotype's orthologue, C-4 unoxygenated 3-ADON, as well as C-4 oxygenated 3-ANIV, accumulated as the end product. A feeding experiment with an ΔFgtri5ΔFgtri3 double gene disruptant, a trichothecene non-producing mutant unable to acetylate C-15 of the trichothecene ring, revealed the importance of the 15-O-acetyl group for efficient C-4 hydroxylation of DON-type trichothecenes. This implies that traditional DON and NIV chemotype diversification is not solely explained by FgTri13, but is also explained by the function of the FgTri8 trichothecene deacetylase gene. None of the crude cell extracts from existing chemotypes showed highly specific C-15 deacetylation activity against 3,15-diacetylnivalenol (3,15-diANIV) without deacetylating C-15 of the C-4 unoxygenated earlier intermediate, 3,15-diacetyldeoxynivalenol. Thus, an unnatural Fusarium trichothecene, 3-ANIV, could only be synthesized as part of a mixture with 3-ADON, unless the esterase encoded by FgTri8 evolves to act on the 15-O-acetyl of 3,15-diANIV with high specificity. We also explain why the transgenic "15-ANIV chemotype", which can be generated through functional inactivation of FgTri7, uses an engineered pathway via 3,15-diANIV, but not 15-ADON, to generate 15-ANIV. Tri genes appear to evolve continuously, and altered functions of trichothecene pathway enzymes result in the generation of new trichothecenes, such as NX-2 and NX-3, which have been recently discovered in field isolates of F. graminearum. As recombination of FgTri8 between existing F. graminearum isolates could give rise to a strain that produces mixtures of DON and NIV-type trichothecenes, it may also be noteworthy to monitor the emergence of a field isolate that invalidates traditional chemotype classification.

In vitro Evaluation of the Safety of Adalimumab for the Eye Under HTLV-1 Infection Status: A Preliminary Study.

Adalimumab (ADA), a fully human monoclonal tumor necrosis factor (TNF)-α antibody, is one of the most widely used biologics in the treatment of inflammatory diseases. However, ADA can exacerbate infectious conditions, induce paradoxical reactions such as inflammation, and cause neoplasia. Human T-cell leukemia virus type 1 (HTLV-1) is an infectious agent that induces inflammation and neoplastic infiltration in the eye. To date, numerous HTLV-1 carriers have been treated with adalimumab to suppress inflammation out of necessity, when standard anti-inflammatory drugs such as steroids and immunosuppressive agents have proven inadequate to control the inflammation. Here, we clarify the safety of adalimumab for the eye under HTLV-1 infectious conditions in vitro. We used the adult retinal pigment epithelial cell line (ARPE)-19 cell line as ocular resident cells, and used MT2 and TL-Om1 as HTLV-1-infected cells. ARPE-19 and MT2/TL-Om1 were co-cultured, and then adalimumab was administered. Production of cytokines and chemokines, TNF-α receptor (TNF-R), HTLV-1 proviral load (PVL), and apoptosis were measured to assess the effects of adalimumab. Contact between ARPE-19 and MT2/TL-Om1 produced inflammatory cytokines such as TNF, interleukin (IL)-6, IL-8 and IL-10, and transduced chemokines such as interferon-inducible protein-10 (IP-10), monocyte chemotactic protein-1 (MCP-1), monokine induced by interferon-γ (MIG), and regulated on activation, normal T cell expressed and secreted (RANTES). No inflammatory cytokines and chemokines were exacerbated by adalimumab. Expression of TNF-R on ARPE-19 and MT2/TL-Om1 cells, HTLV-1 PVLs of MT2/TL-Om1 cells, and cell growth rate and apoptotic rate of ARPE-19 were unaffected by adalimumab. In conclusion, adalimumab does not appear to exacerbate HTLV-1-associated inflammatory conditions in the eye or increase PVL in HTLV-1-infected T cells. These data suggest that adalimumab could be used safely for the eye under HTLV-1 infectious conditions from the perspective of in vitro assessment.

Reduced Toxicity of Trichothecenes, Isotrichodermol, and Deoxynivalenol, by Transgenic Expression of the Tri101 3-O-Acetyltransferase Gene in Cultured Mammalian FM3A Cells.

In trichothecene-producing fusaria, isotrichodermol (ITDol) is the first intermediate with a trichothecene skeleton. In the biosynthetic pathway of trichothecene, a 3-O-acetyltransferase, encoded by Tri101, acetylates ITDol to a less-toxic intermediate, isotrichodermin (ITD). Although trichothecene resistance has been conferred to microbes and plants transformed with Tri101, there are no reports of resistance in cultured mammalian cells. In this study, we found that a 3-O-acetyl group of trichothecenes is liable to hydrolysis by esterases in fetal bovine serum and FM3A cells. We transfected the cells with Tri101 under the control of the MMTV-LTR promoter and obtained a cell line G3 with the highest level of C-3 acetylase activity. While the wild-type FM3A cells hardly grew in the medium containing 0.40 µM ITDol, many G3 cells survived at this concentration. The IC50 values of ITDol and ITD in G3 cells were 1.0 and 9.6 µM, respectively, which were higher than the values of 0.23 and 3.0 µM in the wild-type FM3A cells. A similar, but more modest, tendency was observed in deoxynivalenol and 3-acetyldeoxynivalenol. Our findings indicate that the expression of Tri101 conferred trichothecene resistance in cultured mammalian cells.

Transplacental Gene Delivery (TPGD) as a Noninvasive Tool for Fetal Gene Manipulation in Mice.

Transplacental gene delivery (TPGD) is a technique for delivering nucleic acids to fetal tissues via tail-vein injections in pregnant mice. After transplacental transport, administered nucleic acids enter fetal circulation and are distributed among fetal tissues. TPGD was established in 1995 by Tsukamoto et al., and its mechanisms, and potential applications have been further characterized since. Recently, discoveries of sequence specific nucleases, such as zinc-finger nuclease (ZFN), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) (CRISPR/Cas9), have revolutionized genome editing. In 2019, we demonstrated that intravenous injection of plasmid DNA containing CRISPR/Cas9 produced indels in fetal myocardial cells, which are comparatively amenable to transfection with exogenous DNA. In the future, this unique technique will allow manipulation of fetal cell functions in basic studies of fetal gene therapy. In this review, we describe developments of TPGD and discuss their applications to the manipulation of fetal cells.

Safety of Infliximab for the Eye Under Human T-Cell Leukemia Virus Type 1 Infectious Conditions in vitro.

Use of biologics has been widely advocated for inflammatory diseases recently. Anti-tumor necrosis factor (TNF)-α antibody therapy is reportedly effective against ocular inflammation. However, side effects of TNF-α inhibition have been reported, particularly in the form of exacerbation of infections such as tuberculosis. Paradoxical reactions such as exacerbated inflammation are also well known. Around 20 million humans are infected with human T-cell leukemia virus type 1 (HTLV-1) globally, and this virus can cause adult T-cell leukemia, HTLV-1-associated myelopathy and HTLV-1 uveitis. As for ophthalmic concerns, it has not been identified whether anti-TNF-α antibody stimulates HTLV-1-infected cells and ocular cells to induce HTLV-1 uveitis in HTLV-1 carriers. Here we investigated the effects of anti-TNF-α antibody on ocular status under HTLV-1 infectious conditions using ocular cells and HTLV-1-infected cells in vitro. We used the ARPE-19 human retinal pigment epithelial cell line as ocular cells considered to play an important role in the blood-ocular barrier, and the MT2 HTLV-1-infected cell line. Jurkat cells were used as controls. Infliximab (IFX) was used as an anti-TNF-α antibody to achieve TNF-α inhibition. We evaluated the production of inflammatory cytokines and intercellular adhesion molecule (ICAM)-1, proliferation of ARPE-19, expression of TNF-α receptor (TNF-R) and HTLV-1 proviral DNA, and the percentage of apoptotic ARPE-19. Inflammatory cytokines such as interleukin (IL)-6, IL-8, TNF, and ICAM-1 were significantly elevated through contact between ARPE-19 and MT2. Treatment with IFX tented to inhibit TNF production, although the level of production was low, but changes in IL-6, IL-8, and ICAM-1 remained unaffected. Expression of TNFR was unaltered by IFX treatment. HTLV-1 proviral DNA was not significantly changed with treatment. No change in cell growth rate or apoptotic rate of ARPE-19 was seen with the addition of IFX. In conclusion, IFX did not exacerbate production of inflammatory cytokines, and did not affect expression of TNFR, proliferation of ARPE-19, HTLV-1 proviral load, or apoptosis of ARPE-19. These results suggest that IFX does not exacerbate HTLV-1-related inflammation in the eye and represents an acceptable treatment option under HTLV-1 infectious conditions.

Synthesis and Application of Silver Nanoparticles (Ag NPs) for the Prevention of Infection in Healthcare Workers.

Silver is easily available and is known to have microbicidal effect; moreover, it does not impose any adverse effects on the human body. The microbicidal effect is mainly due to silver ions, which have a wide antibacterial spectrum. Furthermore, the development of multidrug-resistant bacteria, as in the case of antibiotics, is less likely. Silver ions bind to halide ions, such as chloride, and precipitate; therefore, when used directly, their microbicidal activity is shortened. To overcome this issue, silver nanoparticles (Ag NPs) have been recently synthesized and frequently used as microbicidal agents that release silver ions from particle surface. Depending on the specific surface area of the nanoparticles, silver ions are released with high efficiency. In addition to their bactericidal activity, small Ag NPs (<10 nm in diameter) affect viruses although the microbicidal effect of silver mass is weak. Because of their characteristics, Ag NPs are useful countermeasures against infectious diseases, which constitute a major issue in the medical field. Thus, medical tools coated with Ag NPs are being developed. This review outlines the synthesis and utilization of Ag NPs in the medical field, focusing on environment-friendly synthesis and the suppression of infections in healthcare workers (HCWs).