The taxonomic position and the unexpected divergence of the Habu viper, Protobothrops among Japanese subtropical islands.

There are four Habu species currently recognized in Japan: Protobothrops flavoviridis from the Amami Islands and the Okinawa Islands, P. tokarensis from the Tokara Islands, P. elegans from the Yaeyama Islands and Ovophis okinabvensis from the Amami Islands and the Okinawa Islands. To clarify their taxonomic positions, we determined the complete mitochondria genome sequence (approx. 17kb) from two specimens from two different islands each for P. flavoviridis, P. tokarensis and P. elegans as well as one specimen of O. okinavensis and reconstructed the molecular phylogeny of Protobothrops using the published sequences of related species. The maximum likelihood tree showed four major species groups within Protbothrops: Group I consisting of P. cornutus, P. dabieshanensis, P. jerdonii and P. xiangchengensis; Group II consisting of P. flavoviridis and P. tokarensis; Group III consisting of P. maolensis, P. mucrosquamatus and P. elegans; Group IV consisting of P. himalayanus and P. kaubacki. Since we observed an unexpected divergence and the paraphyly of the two samples of P. flavoviridis collected from different islands, Amami-Oshima and Okinawajima within the Group II, we expanded the analysis by increasing the number of P. flavoviridis and P. tokarensis collected from 10 islands: Amami-Oshima (5 specimens), Kakeromajima (4) and Tokunoshima (4) from the Amami Islands, Okinawajima (4), Iheyajima (4), Iejima (4), Tokashikijima (4) and Kumejima (4) from the Okinawa Islands, Kodakarajima (P. tokarensis) (4) and Takarajima (P. tokarensis) (4) from the Tokara Islands. The maximum likelihood tree of the 44 samples replicated the significant divergence of P. flavoviridis between the Amami Clade including Amami-Oshima, Kakeromajima and Tokunoshima and the Okinawa Clade including Okinawajima, Iheyajima, Iejima, Tokashikijima and Kumejima. The Amami Clade also include all specimens from the Tokara Islands currently known as an independent species, P. tokarensis, suggesting the paraphyly of the taxon, P. flavoviridis. In contrast, we observed a distinct lineage of the two specimens from the Yaeyama Islands, supporting the validity of the taxon, P. elegans as an independent species. By MCMC method, we estimated the divergence time between the Amami Clade and the Okinawa Clade to be 6.51MYA, suggesting that the vicariance of the two clades preceded the geological separation of the Amami Islands and the Okinawa Islands (∼1.5MYA). As expected from the limited mobility of terrestrial reptiles including snakes, we observed high genetic divergence in Habu mtDNA among Japanese subtropical island populations.

The Significance of Off-Season Tailor-Made Baseline Measurements in the Assessment of Post-Concussion in University Athletes.

This study aimed to investigate the significance of baseline measurements for amateur team athletes playing contact and collision team sports with a specific focus on the Sports Concussion Assessment Tool (SCAT) to improve concussion management. Symptoms of sports-related concussions (SRCs) can be diverse and long-lasting and include cognitive impairment, sleep disturbances, and vestibular dysfunction. Therefore, comprehensive baseline data are essential to preventing recurrent concussions and secondary injuries. This study was conducted during the 2023 off-season and evaluated the baseline condition of 65 male university rugby players using the SCAT5, which includes self-reported symptoms, and the modified Balance Error Scoring System (mBESS). The athletes were assessed for the presence or absence of SRC, and the mean values were compared using the Mann-Whitney U test. Among the participants, 35.38% (23/65) reported symptoms, with an average of 1.5 ± 2.8 symptoms per player and an average symptom score of 2.66 ± 5.93. In the mBESS, no errors were observed in the tandem stance test; however, 72.31% (47/65) made errors in the single-leg stance test on the non-dominant foot, with an average of 1.7 ± 1.5 errors. Many athletes self-reported symptoms and balance errors, even during asymptomatic periods before experiencing concussion, indicating unresolved issues. In the injury history survey, the baseline evaluations and injury histories of the participants classified into the SRC and non-SRC groups were compared. In the mBESS single-leg stance test (non-dominant foot), 84.21% (32/38) of the SRC group participants made errors, with an average score of 2.13 ± 1.52, whereas 55.55% (15/27) of the non-SRC group participants made errors, with an average score of 1.15 ± 1.35, showing a significant difference (p = 0.007). Additionally, significant differences were observed in the average number of ankle sprains (p = 0.027) and fractures (p = 0.048) between patients with and without a history of SRC. These findings indicate that athletes may have underlying issues even during normal periods before concussion. Moreover, the results highlighted the impact of previous concussions on motor control and injury risk. This underscores the importance of preseason baseline measurements using the SCAT to identify at-risk athletes and implement preventive measures. These findings align with the recommendations of the 6th International Conference on Concussion in Sport and suggest further refinement of concussion assessment tools.

Survey on Sports-Related Concussions among Japanese University Students.

In recent years, head injuries in sports have garnered attention, and in particular, international discussions have been held on the prevention of and response to sports-related concussions (SRCs). The purpose of this study is to investigate past SRCs experienced by university students in Japan, clarify the state and mechanism of such injuries in each sport, and consider the creation of an environment for future SRC prevention and responses. A questionnaire survey on past SRC experience was conducted among 1731 students who belonged to Fukuoka University in Japan and took "sports medicine" classes in 2020. Responses from 1140 students (collection rate: 65.9%) were obtained. According to this survey, it was revealed that 39 students (3.7%) had experienced SRC. The male-female ratio of those who had experienced SRC was 31 males (79.5%) and 8 females (20.5%). Two males had experienced SRC twice. In this study, SRCs were recognized in a variety of sports, not just in a few contact sports. It is necessary to further disseminate education on head injury prevention and SRCs among both athletes and coaches, because SRCs have been frequently recognized in various sports.

Neutralization of hemorrhagic snake venom metalloproteinase HR1a from Protobothrops flavoviridis by human monoclonal antibody.

Human monoclonal antibodies (HuMAbs) against HR1a from Protobothrops (formerly Trimeresurus) flavoviridis venom were obtained by the fusion of SP2/0-Ag14 myeloma cells and spleen cells from KM mice immunized with purified HR1a. The ability of HuMAbs to neutralize the HR1a was determined by in vitro neutralization assay and by neutralization of the hemorrhagic activity. The initial screening of over 300 hybridoma fusion wells resulted in the establishment of 80 HR1a-reactive hybridomas. Of the reactive clones, HuMAb HR1a-7 and HR1a-18 neutralized both proteolytic and hemorrhagic activity of HR1a. Mapping of epitope recognized by the reactive clones was performed by using an ELISA that measured antibody binding to overlapping peptides (15 amino acid peptide offset frameshifted by three residues) covering the metalloproteinase domain sequence of HR1a. HuMAbs HR1a-7 and HR1a-18 neutralized HR1a by reacting with peptides of EQQRYLNNFRFIELV and IVNTLNETYRYL. The three-dimensional structure of HR1a based on a homology modeling predicted that these two epitopes are surface exposed.

The occurrence of HR1b in the venom of the snake Okinawa habu (Protobothrops flavoviridis).

This study describes the first isolation of hemorrhagic metalloproteinase HR1b from the venom of Okinawa habu, and its cDNA cloning. The substrate specificity of isolated HR1b definitely differed from that of HR1a, further supporting the presence of a metalloproteinase distinguishable from HR1a in the venom of Okinawa habu. The deduced amino acid sequence of HR1b showed 99.67% identity with HR1b of Amami habu, with only two amino acid residue replacements.

Discovery of novel [Arg49]phospholipase A2 isozymes from Protobothrops elegans venom and regional evolution of Crotalinae snake venom phospholipase A2 isozymes in the southwestern islands of Japan and Taiwan.

Protobothrops (formerly Trimeresurus) elegans, a Crotalinae snake, inhabits Ishigaki and Iriomote islands of the Sakishima Islands of Japan which are located between Okinawa island of Japan and Taiwan. Two phospholipase A(2) (PLA(2)) isozymes were purified to homogeneity from P. elegans venom and sequenced. This led to a discovery of novel PLA(2) isozymes with Arg at position 49, that is, [Arg(49)]PLA(2) forms, named PeBP(R)-I and PeBP(R)-II. They are polymorphic at position 3, Val for PeBP(R)-I and Ile for PeBP(R)-II. The cDNAs encoding PeBP(R)-I and PeBP(R)-II were cloned. The cDNA encoding an [Asp(49)]PLA(2) named PePLA(2) was also obtained. In contrast to PLA(2) isozymes from Protobothrops genus with 122 amino acid residues, PeBP(R)-I and PeBP(R)-II are composed of 121 amino acid residues due to lack of Pro at position 90. They exhibited necrotic and edema-inducing activities but no hemorrhagic activity was detected. A phylogenetic tree constructed for venom PLA(2) isozymes of Protobothrops genus and of related genera in the southwestern islands of Japan and Taiwan revealed that PeBP(R)-I and PeBP(R)-II of P. elegans are evolutionarily much closer to PmK49PLA(2), a [Lys(49)]PLA(2), from P. mucrosquamatus (Taiwan) than BPI and BPII, both [Lys(49)]PLA(2) forms, from P. flavoviridis (Amami-Oshima and Tokunoshima islands of Japan). Such evolutionary relationships are also seen in neutral [Asp(49)]PLA(2) isozymes from the three Protobothrops species. Thus, P. elegans is the species much closer to P. mucrosquamatus than P. flavoviridis. Their evolutionary distances seem to be well related to geological history of the islands where they have lived. In addition, it was clearly noted that Ovophis okinavensis (Amami-Oshima), which had formerly belonged to the Trimeresurus genus, and Trimeresurus stejnegeri (Taiwan) are the species fairly distant from Protobothrops genus.

Blood flow in snake infrared organs: response-induced changes in individual vessels.

OBJECTIVE: In the past the microkinetics of blood flow in the infrared pit organs of pit vipers has been studied with Doppler flowmetry using various infrared stimuli such as a human hand or soldering iron at various distances, lasers of various wavelengths, etc. Quick-acting variations in blood flow were recorded, and interpreted as a cooling mechanism for avoiding afterimage in the infrared receptors. However, the Doppler measurements provided only the summation of blood flow in a number of vessels covered by the sensing probe, but did not give data on flow in individual vessels. METHODS: In the present work the authors introduced into the bloodstream of Gloydius and Trimeresurus pit vipers fluorescent microspheres labeled with fluorescein isothiocyanate (FITC) contained in a solution of FITC-dextran in physiological saline. They observed the passage of the microspheres through individual pit organ vessels with a fluorescent microscope to which was attached a high-speed video camera and image intensifier. Output of the camera was recorded before, during, and after stimulus with a 810-nm diode laser. Recording was done at 250 frames/s on high-speed video apparatus and downloaded to a hard disk. Disk files were loaded into proprietary software and particles were tracked and average velocities calculated. The data were then tested for significance by ANOVA with post hoc tests. RESULTS: A significant (p<.05) increase in blood velocity was found at the focal point of the stimulus laser, but not anywhere removed from this point. Proximal severing of the pit sensory nerves caused degeneration of the pit receptor terminals and abolished stimulus-induced blood flow changes, but did not affect normal blood flow. CONCLUSIONS: The authors conclude that the receptors themselves are directly and locally controlling the smooth muscle elements of the blood vessels, in response to heating of the receptors by infrared radiation. They speculate that the heavy vascularization constitutes a cooling system for the radiation-encoding receptors, and further that the agent of control may be a volatile neuromediator such as nitric oxide.