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Purpose: Proton stereotactic radiosurgery (PSRS) has emerged as an innovative proton therapy modality aimed at achieving precise dose delivery with minimal impact on healthy tissues. This study explores the dosimetric outcomes of PSRS in comparison to traditional intensity-modulated proton therapy (IMPT) by focusing on cases with small target volumes. A custom-made aperture system designed for proton therapy, specifically tailored to small target volumes, was developed and implemented for this investigation., Methods: A prerequisite mechanical validation through an isocentricity test precedes dosimetric assessments, ensuring the seamless integration of mechanical and dosimetry analyses. Five patients were enrolled in the study, including two with choroid melanoma and three with arteriovenous malformations (AVM). Two treatment plans were meticulously executed for each patient, one utilizing a collimated aperture and the other without. Both plans were subjected to robust optimization, maintaining identical beam arrangements and consistent optimization parameters to account for setup errors of 2 mm and range uncertainties of 3.5%. Plan evaluation metrics encompassing the Heterogeneity Index (HI), Paddick Conformity Index (CI Paddick ), Gradient Index (GI), and the R50% index to evaluate alterations in low-dose volume distribution., Results: The comparative analysis between PSRS and traditional PBS treatment revealed no significant differences in plan outcomes, with both modalities demonstrating comparable target coverage. However, collimated apertures resulted in discernible improvements in dose conformity, dose fall-off, and reduced low-dose volume., Conclusions: This study underscores the advantageous impact of the aperture system on proton therapy, particularly in cases involving small target volumes., (© 2024 The Author(s). Journal of Applied Clinical Medical Physics published by Wiley Periodicals, LLC on behalf of The American Association of Physicists in Medicine.)

Deinagkistrodon acutus is a medically important pitviper inhabiting mainly South China and Taiwan. The hemorrhagic effects of its envenoming are compatible to its venom, which is abundant in metalloproteases (svMPs) and C-type lectin-like proteins. In this study, we investigated geographic variations in the venom of D. acutus collected from Taiwan and four Mainland Chinese provinces: Fujian, Jiangxi, Anhui, and Hunan. The variations were assessed through high-performance liquid chromatography, non-metric multidimensional scaling analysis, gel electrophoresis, and enzyme-linked immunosorbent assay (ELISA) with a monospecific antivenom (DaMAV) generated against the Taiwanese D. acutus venom, and discussed based on venom-protein sequences in databases and literature related to D. acutus venom. Additionally, the cross-reactivity of DaMAV against Crotalus horridus and Calloselasma rhodostoma venoms was investigated. We noted differential abundances of D. acutus venom metalloproteases, C-type lectin-like proteins, and phospholipase A 2 , along with point mutations and selective expression of serine protease isoforms. The ELISA results revealed that the venom from Taiwan was more reactive toward Taiwanese DaMAV than the four Mainland Chinese venoms, consistent with chromatographic profile differences, whereas C. horridus venom presented moderate cross-reactivity with DaMAV. The observed immunoreactivities of these venom with DaMAV can be attributed to the high prevalence of their PIII-svMPs, which are the dominant antigens, and the conservation of PIII-svMP epitopes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Snakebite envenomation is a significant global health issue that requires specific antivenom treatments. In Taiwan, available antivenoms target a variety of snakes, but none specifically target Trimeresurus gracilis, an endemic and protected species found in the high mountain areas of Taiwan. This study evaluated the effectiveness of existing antivenoms against T. gracilis venom, focusing on a bivalent antivenom developed for Trimeresurus stejnegeri and Protobothrops mucrosquamatus (TsPmAV), as well as monovalent antivenoms for Deinagkistrodon acutus (DaAV) and Gloydius brevicaudus (GbAV). Our research involved in vivo toxicity testing in mice and in vitro immunobinding experiments using (chaotropic) enzyme-linked immunosorbent assays, comparing venoms from four pit viper species (T. gracilis, T. stejnegeri, P. mucrosquamatus, and D. acutus) with three types of antivenoms. These findings indicate that TsPmAV partially neutralized T. gracilis venom, marginally surpassing the efficacy of DaAV. In vitro tests revealed that GbAV displayed higher binding capacities toward T. gracilis venom than TsPmAV or DaAV. Comparisons of electrophoretic profiles also reveal that T. gracilis venom has fewer snake venom C-type lectin like proteins than D. acutus, and has more P-I snake venom metalloproteases or fewer phospholipase A2 than G. brevicaudus, T. stejnegeri, or P. mucrosquamatus. This study highlights the need for antivenoms that specifically target T. gracilis, as current treatments using TsPmAV show limited effectiveness in neutralizing local effects in patients. These findings provide crucial insights into clinical treatment protocols and contribute to the understanding of the evolutionary adaptation of snake venom, aiding in the development of more effective antivenoms for human health., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Chuang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: A dual-function phantom designed to quantify the apparent diffusion coefficient (ADC) in different fat contents (FCs) and glass bead densities (GBDs) to simulate the human tissues has not been documented yet. We propose a dual-function phantom to quantify the FC and to measure the ADC at different FCs and different GBDs., Methods: A fat-containing diffusion phantom comprised by 30 glass-bead-containing fat-water emulsions consisting of six different FCs (0, 10, 20, 30, 40, and 50%) multiplied by five different GBDs (0, 0.1, 0.25, 0.5, and 1.0 g/50 mL). The FC and ADC were measured by the "iterative decomposition of water and fat with echo asymmetry and least squares estimation-IQ," IDEAL-IQ, and single-shot echo-planar diffusion-weighted imaging, SS-EP-DWI, sequences, respectively. Linear regression analysis was used to evaluate the relationship among the fat fraction (FF) measured by IDEAL-IQ, GBD, and ADC., Results: The ADC was significantly, negatively, and linearly associated with the FF (the linear slope ranged from -0.005 to -0.017, R 2 = 0.925 to 0.986, all p < 0.001). The slope of the linear relationship between the ADC and the FF, however, varied among different GBDs (the higher the GBD, the lower the slope). ADCs among emulsions across different GBDs and FFs were overlapped. Emulsions with low GBDs plus high FFs shared a same lower ADC range with those with median or high GBDs plus median or lower FFs., Conclusions: A novel dual-function phantom simulating the human tissues allowed to quantify the influence of FC and GBD on ADC., Relevance Statement: The study developed an innovative dual-function MRI phantom to explore the impact of FC on ADC variation that can affect clinical results. The results revealed the superimposed effect on FF and GBD density on ADC measurements., Key Points: • A dual-function phantom made of glass bead density (GBD) and fat fraction (FF) emulsion has been developed. • Apparent diffusion coefficient (ADC) values are determined by GBD and FF. • The dual-function phantom showed the mutual ADC addition between FF and GBD., (© 2024. The Author(s).)

The oral cavity of snakes serves as a habitat for various microorganisms, some of which may include potential zoonotic pathogens posing risks to hosts and causing wound infections in snakebite victims. Clinical studies on snakebite cases in Taiwan have identified specific pathogens, such as Enterococcus faecalis (Gram-positive), Morganella morganii , Aeromonas hydrophila , and Pseudomonas aeruginosa (Gram-negative). However, the prevalence of these bacteria in the oral cavity of wild snakes remains largely unknown. This study investigated the occurrence of these bacteria in six wild snake species ( Naja atra , Bungarus multicinctus , Trimeresurus stejnegeri , Protobothrops mucrosquamatus , Boiga kraepelini , and Elaphe taeniura friesi ) from southern Taiwan, along with factors influencing their presence. Oropharyngeal swab samples were collected from a substantial number of wild-caught snakes ( n = 1104), followed by DNA extraction, polymerase chain reaction, and gel electrophoresis. The band positions of samples were compared with positive and negative controls to determine the presence of target bacteria in each sample. The overall occurrence rates were 67.4% for E. faecalis , 31.5% for M. morganii , 8.2% for A. hydrophila , and 7.7% for P. aeruginosa . Among snake species, B. kraepelini exhibited dominance in E. faecalis (93.4%), A. hydrophila (17.1%), and P. aeruginosa (14.5%), while male N. atra showed dominance in M. morganii (51.3%). The occurrence of E. faecalis was lowest in winter. The results of multiple logistic regression analyses suggest that factors such as species, sex, temperature, season, and coexisting pathogens may have a significant impact on the occurrence of target bacteria. These findings have implications for wildlife medicine and snakebite management.

Snakebite envenomation is a neglected tropical disease. Taiwan, with its subtropical and Southeast Asian environment, provides suitable habitat for several venomous snake species. Trimeresurus stejnegeri, an arboreal pit viper, is the most common cause of venomous snakebite in Taiwan. Trimeresurus stejnegeri envenomation can cause local swelling, occasional ecchymosis, and wound infection. The primary treatment of T. stejnegeri envenomation is the binary antivenom, vacuum freeze-dried F(ab')2 fragments of equine antibodies, against T. stejnegeri and Protobothrops mucrosquamatus. This study aimed to analyze the incidence of post-envenomation wound infections caused by T. stejnegeri based on data collected over a decade from institutions affiliated with the Chang Gung Memorial Hospital in Taiwan. A total of 254 patients were enrolled in this study. Clinical and laboratory data, treatment information, and patient outcomes were extracted from electronic medical records. Wound infection was associated with delay in antivenom initiation (adjusted odds ratio: 3.987; 95% CI: 1.406-11.302). The infection rates were 20.5%, 12.5%, 31.3%, and 48.1% for antivenom administration within 2 hours, 2-4 hours, 4-6 hours, and > 6 hours, respectively. Therefore, early initiation of antivenom treatment (within 6 hours) is recommended. Morganella morganii was cultured from wounds of the patients, whereas Enterobacter cloacae and Enterococcus faecalis were cultured from both the oral cavity of snakes and the wounds of the patients. For post-envenomation patients who develop a local infection, empiric antibiotics such as third-generation cephalosporins, quinolones, and piperacillin/tazobactam are recommended because snakebite wound infections are often polymicrobial in nature.

A venomous snake's oral cavity may harbor pathogenic microorganisms that cause secondary infection at the wound site after being bitten. We collected oral samples from 37 individuals belonging to seven species of wild venomous snakes in Taiwan, including Naja atra (Na), Bungarus multicinctus (Bm), Protobothrops mucrosquamatus (Pm), Trimeresurus stejnegeri (Ts), Daboia siamensis (Ds), Deinagkistrodon acutus (Da), and alpine Trimeresurus gracilis (Tg). Bacterial species were identified using full-length 16S rRNA amplicon sequencing analysis, and this is the first study using this technique to investigate the oral microbiota of multiple Taiwanese snake species. Up to 1064 bacterial species were identified from the snake's oral cavities, with 24 pathogenic and 24 non-pathogenic species among the most abundant ones. The most abundant oral bacterial species detected in our study were different from those found in previous studies, which varied by snake species, collection sites, sampling tissues, culture dependence, and analysis methods. Multivariate analysis revealed that the oral bacterial species compositions in Na, Bm, and Pm each were significantly different from the other species, whereas those among Ts, Ds, Da, and Tg showed fewer differences. Herein, we reveal the microbial diversity in multiple species of wild snakes and provide potential therapeutic implications regarding empiric antibiotic selection for wildlife medicine and snakebite management.

Background: Minimally invasive pancreatic surgery via the multi-port approach has become a primary surgical method for distal pancreatectomy (DP) due to its advantages of lower wound pain and superior cosmetic results. Some studies have applied reduced-port techniques for DP in an attempt to enhance cosmetic outcomes due to the minimally invasive effects. Numerous recent review studies have compared multi-port laparoscopic DP (LDP) and multi-port robotic DP (RDP); most of these studies concluded multi-port RDP is more beneficial than multi-port LDP for spleen preservation. However, there have been no comprehensive reviews of the value of reduced-port LDP and reduced-port RDP., Aim: To search for and review the studies on spleen preservation and the clinical outcomes of minimally invasive DP that compared reduced-port DP surgery with multi-port DP surgery., Methods: The PubMed medical database was searched for articles published between 2013 and 2022. The search terms were implemented using the following Boolean search algorithm: ("distal pancreatectomy" OR "left pancreatectomy" OR "peripheral pancreatic resection") AND ("reduced-port" OR "single-site" OR "single-port" OR "dual-incision" OR "single-incision") AND ("spleen-preserving" OR "spleen preservation" OR "splenic preservation"). A literature review was conducted to identify studies that compared the perioperative outcomes of reduced-port LDP and reduced-port RDP., Results: Fifteen articles published in the period from 2013 to 2022 were retrieved using three groups of search terms. Two studies were added after manually searching the related papers. Finally, 10 papers were selected after removing case reports ( n = 3), non-English language papers ( n = 1), technique papers ( n = 1), reviews ( n = 1), and animal studies ( n = 1). The common items were defined as items reported in more than five papers, and data on these common items were extracted from all papers. The ten studies included a total of 337 patients (females/males: 231/106) who underwent DP. In total, 166 patients (females/males, 106/60) received multi-port LDP, 126 (females/males, 90/36) received reduced-port LDP, and 45 (females/males, 35/10) received reduced-port RDP., Conclusion: Reduced-port RDP leads to a lower intraoperative blood loss, a lower postoperative pancreatic fistula rate, and shorter hospital stay and follow-up duration, but has a lower spleen preservation rate., Competing Interests: Conflict-of-interest statement: The authors have no conflicts of interest to declare., (©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved.)

Trimeresurus gracilis is an endemic alpine pitviper in Taiwan with controversial phylogeny, and its venom proteome remains unknown. In this study, we conducted a proteomic analysis of T. gracilis venom using high-performance liquid chromatography-tandem mass spectrometry and identified 155 toxin proteoforms that belong to 13 viperid venom toxin families. By searching the sequences of trypsin-digested peptides of the separated HPLC fractions against the NCBI database, T. gracilis venom was found to contain 40.3% metalloproteases (SVMPs), 15.3% serine proteases, 6.6% phospholipases A 2 , 5.0% L-amino acid oxidase, 4.6% Cys-rich secretory proteins (CRISPs), 3.2% disintegrins, 2.9% vascular endothelial growth factors (VEGFs), 1.9% C-type lectin-like proteins, and 20.2% of minor toxins, nontoxins, and unidentified peptides or compounds. Sixteen of these proteoforms matched the toxins whose full amino-acid sequences have been deduced from T. gracilis venom gland cDNA sequences. The hemorrhagic venom of T. gracilis appears to be especially rich in PI-class SVMPs and lacks basic phospholipase A 2 . We also cloned and sequenced the cDNAs encoding two CRISP and three VEGF variants from T. gracilis venom glands. Sequence alignments and comparison revealed that the PI-SVMP, kallikrein-like proteases, CRISPs, and VEGF-F of T. gracilis and Ovophis okinavensis are structurally most similar, consistent with their close phylogenetic relationship. However, the expression levels of some of their toxins were rather different, possibly due to their distinct ecological and prey conditions., Competing Interests: The authors declare no conflict of interest.

The cDNAs encoding the Zn +2 -metalloproteases (SVMPs) of Trimeresurus gracilis (abbreviated as Tgc), a pitviper endemic to Taiwan, were cloned from venom glands and sequenced. The amino-acid sequences of five novel SVMPs, including one P-III, three P-II and one P-I class enzymes, were thus deduced and subjected to BLAST-analyses. The P-III enzyme (designated as Tgc-PIII) is structurally most similar to the PIII-SVMPs of New World pitvipers but not similar to the PIII-SVMP of Ovophis okinavensis. Sequence-similarity analysis of 22 homologous PIII-SVMPs reveal three major structural subtypes of the pitviper PIII-SVMPs, which possibly have different substrate specificities. In addition, Tgc-PIII and the PI-class SVMP (named Tgc-MP) were isolated from the venom and verified by mass spectrometry. All the three deduced sequences of PII-SVMPs (Tgc-PIIs) contain an abnormal Zn +2 -binding-site in their catalytic-domain, and an identical "long-disintegrin" domain. The predicted 85-residues disintegrin, gracilisin, bears high similarities to some long-disintegrins of the New-World pitvipers and salmosin3. By BLAST search and comparison, Tgc-MP is 96% similar to okinalysin, the hemorrhagic PI-SVMP of O. okinavensis, rather than any other PI-SVMPs in the databanks. Our results confirm the fast evolution of Tgc-SVMPs as well as their structural similarities to different SVMP-classes of the New-World pitvipers and of O. okinavensis, respectively. The implications of our findings are discussed along with our previous sequence comparisons of venom phospholipases A 2 and ten venom serine proteases of Tgc., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Inn-Ho Tsai reports financial support and equipment, drugs, or supplies were provided by Taiwan Government Ministry of Science and Technology. Tein-Shun Tsai reports financial support was provided by Taiwan Government Ministry of Science and Technology., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Wound infections after venomous snakebites are clinically important. Information regarding the nature and antibiotic susceptibilities of snake oral bacterial flora could support empiric antibiotic therapy. Wild venomous snakes were collected from southern Taiwan: a total of 30 each of Bungarus multicinctus, Naja atra, Protobothrops mucrosquamatus, and Trimeresurus stejnegeri; 3 Deinagkistrodon acutus; and 4 Daboia siamensis. The species and antibiotic susceptibilities of their oral bacteria were determined. Aerobic gram-negative bacteria, especially Pseudomonas aeruginosa and Proteus vulgaris, were the most abundant. Proteus vulgaris were more abundant in B. multicinctus, N. atra, and P. mucrosquamatus than in T. stejnegeri (40%, 43.3%, and 40% vs. 13.3%, respectively). The gram-negative species were less susceptible to first- and second-generation cephalosporins and ampicillin-sulbactam than to third-generation cephalosporins, fluoroquinolones, carbapenems, or piperacillin-tazobactam. The most abundant aerobic gram-positive species cultured was Enterococcus faecalis, which was more abundant in N. atra than in other snakes (p < 0.001) and was highly susceptible to ampicillin, high-level gentamicin, penicillin, teicoplanin, and vancomycin. Bacteroides fragilis and Clostridium species were the most common anaerobic bacteria. The anaerobic organisms were highly susceptible to metronidazole and piperacillin. As a reference for empiric antimicrobial therapy, third-generation cephalosporins, fluoroquinolones, carbapenems, or piperacillin-tazobactam can be initiated in venomous snakebites wound infections.

Trimeresurus gracilis (Tgc) is endemic to Taiwan and shown to be closely related with Ovophis okinavensis by previous phylogenetic analyses, but their taxonomic status remain controversial. Here, we cloned and sequenced ten of its venom serine-proteases (designated as Tgc-vSPs). All the Tgc-vSPs conserve the catalytic triads, six appear to be kallikrein-like (KNs) and four are plasminogen-activator homologs (PAHs and PAs). They are studied under four structural categories: (1) highly similar Tgc-KN1, Tgc-KN2 and Tgc-KN3, with four predicted N-glycosylation sites; (2) Tgc-KN4, with a single N -glycosylation site; (3) Tgc-KN5 and Tgc-KN6, with two distinct N-glycosylation sites; (4) Tgc-PAH1/PAH2, TgcPA3, and Tgc-PA4, with two conserved N-glycosylation sites. Additionally, Tgc-KN1, Tgc-KN4 and Tgc-PAH1 were purified by reversed-phase HPLC and identified by peptide-mass-fingerprinting. Results of BLAST and sequence alignments reveal that Tgc-KN1∼3 and Tgc-KN6 are most like the vSPs of rattlesnakes, while the sequences of Tgc-KN4, KN5 and Tgc-PAH1/PAH2 match closely to the partial sequences of three O. okinavensis vSPs. Thus, our results reveal non-overlapping similarities of Tgc-vSPs to the O. okinavensis vSPs and vSPs of the New World pitvipers. In addition, molecular phylogenetic analyses of the plasminogen-activator like vSPs reveal separate evolution of two clusters of the enzymes with distinct functions., (Copyright © 2021 Elsevier Ltd. All rights reserved.)