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.

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.

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.

Molecular Diversity of Linear Peptides Revealed by Transcriptomic Analysis of the Venom Gland of the Spider Lycosa poonaensis.

Spider venom is a complex mixture of bioactive components. Previously, we identified two linear peptides in Lycosa poonaensis venom using mass spectrometric analysis and predicted the presence of more linear peptides therein. In this study, a transcriptomic analysis of the L. poonaensis venom gland was conducted to identify other undetermined linear peptides in the venom. The results identified 87 contigs encoding peptides and proteins in the venom that were similar to those in other spider venoms. The number of contigs identified as neurotoxins was the highest, and 15 contigs encoding 17 linear peptide sequences were identified. Seven peptides that were representative of each family were chemically synthesized, and their biological activities were evaluated. All peptides showed significant antibacterial activity against Gram-positive and Gram-negative bacteria, although their selectivity for bacterial species differed. All peptides also exhibited paralytic activity against crickets, but none showed hemolytic activity. The secondary structure analysis based on the circular dichroism spectroscopy showed that all these peptides adopt an amphiphilic α-helical structure. Their activities appear to depend on the net charge, the arrangement of basic and acidic residues, and the hydrophobicity of the peptides.

Successful limb salvage beyond the golden time following blunt traumatic open complete transection of the femoral artery and vein in a patient with cardiac arrest: a case report.

BACKGROUND: Open complete transection of the femoral artery and vein following blunt trauma is extremely rare. Furthermore, even if the patient has been successfully resuscitated, it is sometimes difficult in most patients to preserve the injured limb, especially after damage control resuscitation. We report a case of open complete transection of the femoral artery and vein secondary to high-energy blunt trauma and a successful limb preservation treatment strategy. CASE PRESENTATION: A 57-year-old Asian man was transferred to hospital after having fallen from the 15th floor of a condominium. The patient was in cardiac arrest at the scene, but was successfully resuscitated by emergency medical services staff. On arrival, the patient's hemodynamics were completely collapsed with active external bleeding from the thigh, so we immediately started resuscitation including activation of massive transfusion protocol and temporarily ligated the transected proximal superficial femoral artery, deep femoral artery just distal after branching lateral femoral circumflex artery and the superficial femoral vein. Following radiological findings showing a potential pelvic fracture with active bleeding, we also performed retroperitoneal packing in the resuscitation room and moved the patient to the angiography room for transcatheter arterial embolization. The patient's consciousness was preserved and perfusion of the injured limb was barely maintained after his hemodynamics were adequately stabilized. As we detected weak perfusion of the lower limb via a potential collateral flow from the lateral femoral circumflex artery branches from deep femoral artery by pulse doppler of the dorsal pedis artery, we decided to reconstruct superficial femoral artery and vein at 24 h after injury using great saphenous vein bypass grafts. The patient was transferred to a rehabilitation hospital with good neurological and limb outcome after hospitalization for 52 days. CONCLUSION: We successfully preserved the patient's lower limb after cardiac arrest and complete transection of the femoral artery and vein and achieved a good neurological outcome. Even if a femoral artery needs to be ligated temporarily, careful observation and assessment should be performed so as not to lose the chance to salvage the limb even during damage control resuscitation.

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.

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.

Identification of an antiviral component from the venom of the scorpion Liocheles australasiae using transcriptomic and mass spectrometric analyses.

Scorpion venom contains a variety of biologically active peptides. Among them, neurotoxins are major components in the venom, but it also contains peptides that show antimicrobial activity. Previously, we identified three insecticidal peptides from the venom of the Liocheles australasiae scorpion, but activities and structures of other venom components remained unknown. In this study, we performed a transcriptome analysis of the venom gland of the scorpion L. australasiae to gain a comprehensive understanding of its venom components. The result shows that potassium channel toxin-like peptides were the most diverse, whereas only a limited number of sodium channel toxin-like peptides were observed. In addition to these neurotoxin-like peptides, many non-disulfide-bridged peptides were identified, suggesting that these components have some critical roles in the L. australasiae venom. In this study, we also isolated a component with antiviral activity against hepatitis C virus using a bioassay-guided fractionation approach. By integrating mass spectrometric and transcriptomic data, we successfully identified LaPLA2-1 as an anti-HCV component. LaPLA2-1 is a phospholipase A2 having a heterodimeric structure that is N-glycosylated at the N-terminal region. Since the antiviral activity of LaPLA2-1 was inhibited by a PLA2 inhibitor, the enzymatic activity of LaPLA2-1 is likely to be involved in its antiviral activity.

Long-term outcomes of surgical rib fixation in patients with flail chest and multiple rib fractures.

BACKGROUND: Recently, interest has increased in surgical fixation for severe thoracic wall injury with good short-term outcomes. However, few reports have evaluated long-term outcomes or complications. This study aimed to assess long-term quality of life and implant-related complications after rib fixation for flail chest and multiple rib fractures. METHODS: We interviewed patients who had undergone rib fixation from January 2014 to December 2019 about their current ability to work and their usual life. RESULTS: Twenty-two patients underwent rib fixation during the study period. Two patients with flail chest had already died after the surgery due to senescence; thus, follow-up information was obtained from 20 patients (91%), with a follow-up duration of 47.5 (IQR 22-58) months. The most undesirable event occurring during the study period was irritation caused by a palpable plate (n=2, 10%), probably due to the thin skin of patients over 70 years old. Eighteen patients were able to return to their usual life or same work as in the premorbid state with no complaints. Two patients are still undergoing rehabilitation due to concomitant extremities fractures. The median EQ-5D-5L index score was 0.89 (IQR 0.84-0.93). There were no implant-related complications requiring plate explantation. DISCUSSION: We concluded that rib fixation offers good long-term benefits, with the ability of the patient with flail chest or multiple rib fractures to return to activity in the premorbid state. Elderly patients especially with thin, soft tissue may complain of irritation caused by the plate and should be informed of this prior to surgery. LEVEL OF EVIDENCE: Level IV therapeutic care/management.

How to deploy a uniform and simplified acute-phase management strategy for traumatic pancreatic injury in any situation.

AIM: Management of traumatic pancreatic injury is challenging, and mortality and morbidity remain high. Because pancreatic injury is uncommon and strong recommendations for pancreatic injury management are lacking, management is primarily based on institutional practices. We propose our strategy of pancreatic injury management. METHODS: We retrospectively reviewed patients with pancreatic injury and evaluated our strategy and outcomes. RESULTS: From January 2013 to December 2019, 18 patients were included with traumatic pancreatic injury. The median Injury Severity Score was 22 (25-75% interquartile range, 17-34) and probability of survival was 0.87 (25-75% interquartile range, 0.78-0.93). Patients were grouped according to the American Association for the Surgery of Trauma injury grades: grade I, n = 3 (16.7%); II, n = 6 (33.3%); III, n = 7 (38.9%); and IV, n = 2 (11.1%). All patients underwent endoscopic pancreatic ductal evaluation within 1-2 days after admission. Abbreviated surgery because of hemodynamic instability and subsequent open abdominal management were undertaken in one patient with pancreas head injury and two patients with pancreas body/tail injury. Management was by laparotomy for closed suction drain insertion with main ductal endoscopic drainage in six patients, endoscopic ductal drainage only in six patients, and distal pancreatectomy with closed suction drainage and endoscopic drainage in five patients. One patient with grade I injury underwent observation only. Median length of closed suction drainage was 12 days and that of hospital stay was 36 days. The observed mortality during the study period was 0%. Late formation of pseudo-pancreatic cyst was observed in two patients (11.1%). CONCLUSION: Our uniform, simplified strategy offers good outcomes for any pancreatic injury site and any concomitant injuries, even in hemodynamically unstable patients.

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.

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.

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.

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 Insecticidal Toxins from the Venom of the North African Scorpion, Buthacus leptochelys.

Various bioactive peptides have been identified in scorpion venom, but there are many scorpion species whose venom has not been investigated. In this study, we characterized venom components of the North African scorpion, Buthacus leptochelys, by mass spectrometric analysis and evaluated their insect toxicity. This is the first report of chemical and biological characterization of the B. leptochelys venom. LC/MS analysis detected at least 148 components in the venom. We isolated four peptides that show insect toxicity (Bl-1, Bl-2, Bl-3, and Bl-4) through bioassay-guided HPLC fractionation. These toxins were found to be similar to scorpion α- and ß-toxins based on their N-terminal sequences. Among them, the complete primary structure of Bl-1 was determined by combination of Edman degradation and MS/MS analysis. Bl-1 is composed of 67 amino acid residues and crosslinked with four disulfide bonds. Since Bl-1 shares high sequence similarity with α-like toxins, it is likely that it acts on Na+ channels of both insects and mammals.

Stress Fracture of the Hamate Hook in a Water Polo Player.

Hamate hook fractures are usually caused by direct trauma while using a tennis racket or a baseball bat. We report stress fracture of the hamate hook in a water polo player without any specific trauma. We consider that the stress fracture occurred via indirect mechanisms through the flexor tendons. Strong ulnar deviation of the wrist during ball release and strong grip on a ball with outstretched fingers, which are unique to water polo, were the likely causes of the stress fracture of the hamate hook.

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.