Transcriptionally linked simultaneous overexpression of P450 genes for broad-spectrum herbicide resistance.

Broad-spectrum herbicide resistance (BSHR), often linked to weeds with metabolism-based herbicide resistance, poses a threat to food production. Past studies have revealed that overexpression of catalytically promiscuous enzymes explains BSHR in some weeds; however, the mechanism of BSHR expression remains poorly understood. Here, we investigated the molecular basis of high-level resistance to diclofop-methyl in BSHR late watergrass (Echinochloa phyllopogon) found in the United States, which cannot be solely explained by the overexpression of promiscuous cytochrome P450 monooxygenases CYP81A12/21. The BSHR late watergrass line rapidly produced 2 distinct hydroxylated diclofop acids, only 1 of which was the major metabolite produced by CYP81A12/21. RNA-seq and subsequent reverse transcription quantitative PCR (RT-qPCR)-based segregation screening identified the transcriptionally linked overexpression of a gene, CYP709C69, with CYP81A12/21 in the BSHR line. The gene conferred diclofop-methyl resistance in plants and produced another hydroxylated diclofop acid in yeast (Saccharomyces cerevisiae). Unlike CYP81A12/21, CYP709C69 showed no other herbicide-metabolizing function except for a presumed clomazone-activating function. The overexpression of the 3 herbicide-metabolizing genes was also identified in another BSHR late watergrass in Japan, suggesting a convergence of BSHR evolution at the molecular level. Synteny analysis of the P450 genes implied that they are located at mutually independent loci, which supports the idea that a single trans-element regulates the 3 genes. We propose that transcriptionally linked simultaneous overexpression of herbicide-metabolizing genes enhances and broadens the metabolic resistance in weeds. The convergence of the complex mechanism in BSHR late watergrass from 2 countries suggests that BSHR evolved through co-opting a conserved gene regulatory system in late watergrass.

Characterization of 2 linear peptides without disulfide bridges from the venom of the spider Lycosa poonaensis (Lycosidae).

Spider venom is a complex mixture of bioactive components, in which peptides play an important role by showing neurotoxicity or cytotoxicity. Disulfide-rich peptides are major components in the venom, but linear peptides without disulfide bridges are also present and often show antimicrobial activity. In this study, we analyzed the venom of the spider Lycosa poonaensis (Lycosidae) to find novel antimicrobial peptides using mass spectrometry. The result revealed that 120 out of 401 detected components were nondisulfide-bridged peptides. From them, the sequence of 2 peptides (lyp2370 and lyp1987) were determined by MS/MS analysis. The biological activity test revealed that lyp2370 has only weak antibacterial activity. On the other hand, lyp1987, which is identical to M-lycotoxin-Ls3b from the Lycosa singoriensi venom, showed significant antibacterial activity. The weak activity of lyp2370 was found to be due to the presence of a Glu residue on the hydrophilic face of its amphipathic α-helical structure.

COVID-19-associated pulmonary aspergillosis in a Japanese man: A case report.

CAPA (COVID-19 associated pulmonary aspergillosis) is an important complication of COVID-19. It has been reported that the incidence of CAPA is as high as 19%-33% worldwide. However, its onset has not been reported in Japan. A 72-year-old Japanese man was diagnosed with COVID-19 and was transferred to our hospital due to deterioration of respiratory condition. Treatment with remdesivir, dexamethasone (DEXA), and antibiotics was performed under mechanical ventilation. Although the condition improved temporarily, a new shadow appeared in the lung, and Aspergillus fumigatus was cultured from sputum. The patient was clinically diagnosed with CAPA and treated with voriconazole. However, his progress deteriorated and he died. High-risk COVID-19 patients should be tested for Aspergillus to ensure early diagnosis of CAPA.

A Fluorescent Compound from the Exuviae of the Scorpion, Liocheles australasiae.

Most scorpions fluoresce under UV light. To date, two types of fluorescent compounds have been identified in scorpions, but it has been assumed that other unknown compounds may be responsible for the fluorescence. In this study, we isolated a fluorescent compound from the exuviae of the scorpion Liocheles australasiae identified as a macrocyclic diphthalate ester with a molecular mass of 496.2 Da. The same compound was also detected in extracts from four other scorpion species. This suggests that this compound is shared by multiple scorpion species, although its contribution to the cuticle fluorescence may be relatively small.

Effects of a pyroglutamyl pentapeptide isolated from fermented barley extract on atopic dermatitis-like skin lesions in hairless mouse.

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by pruritic and eczematous skin lesions. The skin of AD patients is generally in a dried condition. Therefore, it is important for AD patients to manage skin moisturization. In this study, we examined the effects of orally administered fermented barley extract P (FBEP), which is prepared from a supernatant of barley shochu distillery by-product, on stratum corneum (SC) hydration and transepidermal water loss (TEWL) in AD-like lesions induced in hairless mice using 2,4,6-trinitrochlorobenzene. Oral administration of FBEP increased SC hydration and decreased TEWL in the dorsal skin of this mouse model. Further fractionation of FBEP showed that a pyroglutamyl pentapeptide, pEQPFP comprising all -L-form amino acids, is responsible for these activities. These results suggested that this pyroglutamyl pentapeptide may serve as a modality for the treatment of AD.

Selective Cytotoxicity of Staphylococcal α-Hemolysin (α-Toxin) against Human Leukocyte Populations.

Staphylococcus aureus produces a variety of exoproteins that interfere with host immune systems. We attempted to purify cytotoxins against human leukocytic cells from the culture supernatant of S. aureus by a combination of ammonium sulfate precipitation, ion-exchange chromatography on a CM-cellulose column and HPLC on a Mono S 5/50 column. A major protein possessing cytotoxicity to HL60 human promyelocytic leukemia cells was purified, and the protein was identified as α-hemolysin (Hla, α-toxin) based on its molecular weight (34 kDa) and N-terminal amino acid sequence. Flow cytometric analysis suggested differential cytotoxicity of Hla against different human peripheral blood leukocyte populations. After cell fractionation with density-gradient centrifugation, we found that peripheral blood mononuclear cells (PBMCs) were more susceptible to Hla than polymorphonuclear leukocytes. Moreover, cell surface marker analysis suggested that Hla exhibited slightly higher cytotoxicity against CD14-positive PBMCs (mainly monocytes) than CD3- or CD19-positive cells (T or B lymphocytes). From these results, we conclude that human leukocytes have different susceptibility to Hla depending on their cell lineages, and thereby the toxin may modulate the host immune response.

Isolation and characterization of the insecticidal, two-domain toxin LaIT3 from the Liocheles australasiae scorpion venom.

A novel insecticidal peptide (LaIT3) was isolated from the Liocheles australasiae venom. The primary structure of LaIT3 was determined by a combination of Edman degradation and MS/MS de novo sequencing analysis. Discrimination between Leu and Ile in MS/MS analysis was achieved based on the difference in side chain fragmentation assisted by chemical derivatization. LaIT3 was determined to be an 84-residue peptide with three intrachain disulfide bonds. The sequence similarity search revealed that LaIT3 belongs to the scorpine-like peptides consisting of two structural domains: an N-terminal α-helical domain and a C-terminal cystine-stabilized domain. As observed for most of the scorpine-like peptides, LaIT3 showed significant antibacterial activity against Escherichia coli, which is likely to be caused by its membrane-disrupting property.

Chemical synthesis of a two-domain scorpion toxin LaIT2 and its single-domain analogs to elucidate structural factors important for insecticidal and antimicrobial activities.

Scorpion venom contains various bioactive peptides. Among them, peptides having two different structural domains constitute a toxin family known as ß-KTx or scorpine-like peptides. These peptides consist of an α-helical structure in the N-terminal region and a cysteine-stabilized structure in the C-terminal region. This unique structure of ß-KTx peptides contributes to their diverse biological functions, but the importance of each domain for their activities is not fully understood. LaIT2 is a ß-KTx peptide isolated from the venom of the scorpion Liocheles australasiae, which shows both insecticidal and antimicrobial activities. In this study, we chemically synthesized full-length LaIT2 using a native chemical ligation technique as well as its N-terminally or C-terminally truncated single-domain analogs to evaluate structural factors important for the activities. Biological evaluation of these peptides revealed that the N-terminal α-helical domain of LaIT2 is essential for the expression of both insecticidal and antibacterial activities. This suggests that the disruption of membrane structures largely accounts for the biological activities of LaIT2.

Isolation, structural identification and biological characterization of two conopeptides from the Conus pennaceus venom.

A novel anti-mollusk conopeptide pn4c was isolated from the Conus pennaceus venom by repeated HPLC fractionation based on the activity against freshwater snails. The primary structure of pn4c was determined by the mass spectrometric de novo sequencing analysis. In addition, pn3a was isolated from the same fraction containing pn4c, as a peptide with unknown functions.

Early signaling events induced by the peptide elicitor PIP-1 necessary for acetosyringone accumulation in tobacco cells.

A peptide elicitor PIP-1 induces defense-related secondary metabolites such as phytoalexin capsidiol in tobacco cells. In this study, we identified one of other metabolites induced by PIP-1 as acetosyringone. Unlike capsidiol accumulation that requires long-term stimulation with PIP-1, acetosyringone was induced by short-term stimulation with PIP-1. The importance of NADPH oxidase in the acetosyringone induction was also demonstrated.

Characterization of the venom of the vermivorous cone snail Conus fulgetrum.

Over 200 components with molecular mass ranging mainly from 400 to 4000 Da were characterized from the venom of the vermivorous cone snail Conus fulgetrum that inhabit Egyptian Red Sea. One major component having a molecular mass of 2946 Da was purified by HPLC, and its primary structure was determined by a combination of Edman degradation and MS/MS analysis.

Chemical synthesis of La1 isolated from the venom of the scorpion Liocheles australasiae and determination of its disulfide bonding pattern.

La1 is a 73-residue cysteine-rich peptide isolated from the scorpion Liocheles australasiae venom. Although La1 is the most abundant peptide in the venom, its biological function remains unknown. Here, we describe a method for efficient chemical synthesis of La1 using the native chemical ligation (NCL) strategy, in which three peptide components of less than 40 residues were sequentially ligated. The peptide thioester necessary for NCL was synthesized using an aromatic N-acylurea approach with Fmoc-SPPS. After completion of sequential NCL, disulfide bond formation was carried out using a dialysis method, in which the linear peptide dissolved in an acidic solution was dialyzed against a slightly alkaline buffer to obtain correctly folded La1. Next, we determined the disulfide bonding pattern of La1. Enzymatic and chemical digests of La1 without reduction of disulfide bonds were analyzed by liquid chromatography/mass spectrometry (LC/MS), which revealed two of four disulfide bond linkages. The remaining two linkages were assigned based on MS/MS analysis of a peptide fragment containing two disulfide bonds. Consequently, the disulfide bonding pattern of La1 was found to be similar to that of a von Willebrand factor type C (VWC) domain. To our knowledge, this is the first report of the experimental determination of the disulfide bonding pattern of peptides having a single VWC domain as well as their chemical synthesis. La1 synthesized in this study will be useful for investigation of its biological role in the venom.

Sonographic evaluation of effects of the volar plate on trigger finger.

BACKGROUND: We evaluated trigger fingers ultrasonographically and clarified differences between fingers with and without continuous locking or snapping symptoms according to the thicknesses of the A1 pulley, flexor tendon and volar plate. METHODS: We evaluated 26 trigger fingers, divided into two groups: Group 1, 14 fingers with locking or snapping; and Group 2, 12 fingers without such symptoms. We also evaluated 26 contralateral fingers as controls (Control 1 and 2 groups). We compared each group to the respective control group according to thickness of the A1 pulley and volar plate, and cross-sectional area of the flexor tendon. In addition, nine fingers with locking or snapping and treated using corticosteroid injection were evaluated according to symptoms and sonographic findings 3-4 weeks after treatment. RESULTS: Thickness of the A1 pulley and cross-sectional area of the flexor tendon were greater in both Groups 1 and 2 than in controls. Thickness of the volar plate was greater in Group 1 than in Control 1, although no significant difference was seen between Group 2 and Control 2. In Group 1, eight of the nine fingers showed an alleviation of locking or snapping symptoms with corticosteroid injection, and sonographic findings showed that thickness of the volar plate was significantly decreased with corticosteroid injection, in addition to reduced thickness of the A1 pulley. CONCLUSION: In addition to thickening of the A1 pulley, thickening of the volar plate may represent an important contributor to continuous snapping or locking symptoms.

Rare Case of Open Comminuted Scaphoid Fracture Dislocation: Treatment With Free Bone Graft and Locking Plate.

To the best of our knowledge, there are no reports in the literature of an open comminuted scaphoid fracture dislocation. We present such a rare case. The case report illustrates the case of a 58-year-old right-handed press operator who injured his left wrist when his hand got caught in a press machine. He received initial treatment at another hospital and was subsequently referred to our hospital. Eight days after the injury, surgery was performed under the brachial plexus block. Successful bone fusion was achieved through volar locking plate fixation, primary free bone grafting from the radius, and Kirschner wire fixation. Our case report may be a valuable resource for the treatment of similar injuries.

Chemical synthesis and functional characterization of LaIT3, an insecticidal two-domain peptide in Liocheles australasiae venom.

LaIT3, belonging to the ß-KTx family, is an insecticidal peptide in the venom of the Liocheles australasiae scorpion. Peptides in the family consist of two structural domains: an N-terminal domain with an α-helical structure common to antimicrobial peptides and a C-terminal domain with a structure stabilized by three disulfide bonds common to ion-channel blocking peptides. However, the contribution of each domain of LaIT3 to its activity remained unknown. In addition, some peptidic components are known to be enzymatically cleaved in the venom, which generates partial peptides. In our study, we searched for partial peptides of LaIT3 using LC/MS analysis and found peptides generated by cleavage at the central region of LaIT3. We subsequently synthesized full-length LaIT3 and its partial peptides to evaluate their insecticidal activity. The results, showing that only full-length LaIT3 is active, indicate that the insecticidal activity of LaIT3 depends on the presence of both N-terminal and C-terminal domains. Furthermore, LaIT3 did not exhibit the cytolytic activity against insect cells and showed only weak antibacterial activity. These findings suggest that its action is not due to a simple membrane disruption effect but instead due to actions on specific target molecules, including ion channels.

Quantitative structure-activity relationship of 2,6-dimethoxy-N-(3-(4-substituted phenyl)isoxazol-5-yl)benzamide for the inhibition of chitin synthesis.

Previously, we found that 5-(2,6-dimethoxybenzoylamino)-3-phenylisoxazoles (IOXs) inhibit chitin synthesis in the cultured integument of Chilo suppressalis. In this study, IOXs with various substituents at the para-position of the 3-phenyl ring were synthesized, and the concentrations required to inhibit chitin synthesis to 50% (IC50) were determined for all compounds. The introduction of halogens-such as F, Cl, and Br-and small alkyls-such as Me, Et, Pr, and n-Bu-at the 3-phenyl ring slightly enhanced the activity. However, the activity decreased drastically with the introduction of NO2, CF3, and t-Bu. The quantitative analysis of the substituent effect at the 3-phenyl ring on chitin-synthesis inhibition using the Hansch-Fujita method showed that the hydrophobic substituent with the optimum value was favored for the activity, but the bulky substituent in terms of E s was detrimental to the activity.

Isolation and characterization of an anti-insect ß-toxin from the venom of the scorpion Isometrus maculatus.

Im-3 was isolated from the venom of the scorpion Isometrus maculatus through several steps of HPLC fractionation based on the insect paralytic activity. Injecting Im-3 into crickets induced paralysis, but no toxicity was apparent in mice after an intracerebroventricular injection. Im-3 shares sequence similarity to scorpion ß-toxins that specifically affect insect sodium channels.

Combined computed tomography and C-arm resuscitation room system (CTCARM) is associated with decreased time to definitive hemostasis and reduces preperitoneal pelvic packing maneuvers in severe pelvic trauma.

Objectives: Severe pelvic fracture concomitant with massive bleeding is potentially lethal, and intervention for hemorrhage control still depends on institutional supplies. With the recent installation of a CT and C-arm combined resuscitation room system (CTCARM) for treatment of trauma patients in our institution, the strategic process and options for hemorrhage control after pelvic fracture have changed. We retrospectively reviewed the procedures we performed and their outcomes. Methods: The CTCARM was installed in our trauma resuscitation room in April 2020. Patients who were diagnosed as having pelvic fracture and underwent interventional radiology for hemorrhage control within 2.5 hours after arrival were compared before and after CTCARM installation. We reviewed the time process for hemorrhage control, treatment options performed, blood products used and their outcomes. Results: Included in this study were 56 patients treated between 2016 and 2022, of whom 36 patients were treated before (original group) and 20 patients after CTCARM installation (CTCARM group). Patient characteristics and vital signs at admission were not statistically different. Preperitoneal pelvic packing was performed significantly more frequently in the original group (p<0.01), whereas resuscitative endovascular balloon occlusion of the aorta use was much more frequent in the CTCARM group (p=0.02). Although the times from admission to first angiography (p=0.014) and to complete hemostasis (p=0.02) were significantly shorter in the CTCARM group, mortality was not statistically different. Four preventable trauma deaths occurred in the original group, but there were none in the CTCARM group. Six unexpected survivors were observed in the original group and four in the CTCARM group. Conclusions: Although the CTCARM had no direct effects on patient mortality for now, it has allowed us to accelerate the treatment time process, shorten preperitoneal pelvic packing procedural time, and potentially avoid subsequent preventable trauma deaths. Level of evidence: Level IV.

Isolation and characterization of a novel non-selective ß-toxin from the venom of the scorpion Isometrus maculatus.

Scorpion venom is composed of a number of peptides, many of which show neurotoxicity. The Isometrus maculatus scorpion, belonging to the Buthidae family, is found in many tropical regions, including the southern islands of Japan, but there have been no reports on the isolation of toxins from its venom affecting sodium channels. We isolated in this study a novel toxin, Im-2, from the I. maculatus venom. Im-2 induced paralysis in crickets after injecting 20 µg/g of body weight. Im-2 also induced lethality in mice after an intracerebroventricular injection, indicating that Im-2 had non-selective toxicity between insects and mammals. Im-2 consists of 68 amino acids cross-linked by 4 disulfide bonds, and has sequence similarity to scorpion ß-toxins that have been reported to affect the sodium channels of both insects and mammals. The toxic symptoms caused by Im-2 suggest that it acted on the nervous system and shared the action mechanism(s) with sequence-homologous ß-toxins.