Sonodynamic therapy (SDT): a novel strategy for cancer nanotheranostics

Sonodynamic therapy (SDT) is a promising non-invasive therapeutic modality. Compared to photo-inspired therapy, SDT provides many opportunities and benefits, including deeper tissue penetration, high precision, less side effects, and good patient compliance. Thanks to the facile engineerable nature of nanotechnology, nanoparticles-based sonosensitizers exhibit predominant advantages, such as increased SDT efficacy, binding avidity, and targeting specificity. This review aims to summarize the possible mechanisms of SDT, which can be expected to provide the theoretical basis for SDT development in the future. We also extensively discuss nanoparticle-assisted sonosensitizers to enhance the outcome of SDT. Additionally, we focus on the potential strategy of combinational SDT with other therapeutic modalities and discuss the limitations and challenges of SDT toward clinical applications.     

Diabetes-associated complications in Spontaneously Diabetic Torii fatty rats.

The Spontaneously Diabetic Torii (SDT) fatty rat, established by introducing the fa allele of the Zucker fatty rat into the SDT rat genome, is a new model of obese type 2 diabetes. The SDT-fa/fa (SDT fatty) rat shows overt obesity, and hyperglycemia and hyperlipidemia are observed at a young age as compared with the SDT-+/+ (SDT normal) rat. However, the features of the diabetic complications in the SDT fatty rat have not been reported. In the present study, the incidence and the progression of diabetic complications in the SDT fatty rat were examined, and compared with those of the SDT normal rat. Renal function parameters, such as blood urea nitrogen, urine volume and urinary protein, increased from 4 weeks of age in the SDT fatty rat, and pathological findings in the renal tubule were observed from 8 weeks. Furthermore, cataract was observed in the SDT fatty rat from 8 weeks of age, and prolongation of peak latencies on electroretinograms was observed at 16 and 24 weeks of age. On the other hand, in the SDT normal rat, renal or ocular changes were observed from 24 weeks of age. With early incidence of diabetes mellitus, diabetes-associated complications in the SDT fatty rat were seen at younger ages than those in the SDT normal rat. In conclusion, the SDT fatty rat is expected to be a useful model for the analysis of diabetic complications and the evaluation of drugs related to metabolic diseases.     

自噬在声动力疗法抑制C6胶质瘤细胞增殖中的作用

目的:探讨自噬在血卟啉单甲醚(Hematoporphyrin monomethyl ether,HMME)介导的声动力疗法(Sonodynamic therapy,SDT)抑制C6胶质瘤细胞增殖中的作用。方法:选取对数期生长的C6胶质瘤细胞并随机分为四组:对照组(未予处理)、超声组(单独超声照射)、HMME组(单独加入HMME)、SDT组(超声照射+HMME)。透射电镜观察SDT处理的C6胶质瘤细胞中自噬体数量的改变。应用qRT-PCR和免疫印迹分析SDT处理对C6胶质瘤细胞中的LC3、Beclin1、Bcl-2 m RNA及蛋白表达水平的影响。MTT检测C6胶质瘤细胞的活力变化。结果:透射电子显微镜显示SDT组自噬体数量较对照组明显增多。SDT组C6胶质瘤细胞中微管相关蛋白1轻链3 (Microtubule associated protein 1 light chain 3, LC3)、Beclin1 m RNA和蛋白水平高于对照组,B细胞淋巴瘤-2(B cell lymphoma-2, Bcl-2) m RNA和蛋白水平低于对照组。与对照组相比,SDT组C6胶质瘤细胞存活率从0 h至6 h逐渐下降,从12 h至72 h逐渐升高。3-甲基腺嘌呤(3-Methyladenine,3-MA)+SDT、氯喹(Chloroquine,CQ)+SDT处理后C6胶质瘤细胞存活率较SDT组明显降低。结论:SDT可能通过诱导自噬抑制C6胶质瘤细胞增殖。     

Effect of insulin therapy on renal changes in spontaneously diabetic Torii rats.

The spontaneously diabetic Torii (SDT) rat has recently been established as an animal model of non-obese type 2 diabetes, in which ocular complications severe occur. However, the function and morphological features of the diabetic renal lesions in SDT rats have not been reported in detail. Therefore, we evaluated changes over time in renal lesions in SDT rats. In addition, SDT rats were treated with insulin to observe whether these renal complications are caused by hyperglycemia. Renal functional parameters and renal lesions were monitored in SDT rats from 8 to 68 weeks of age. Sprague-Dawley (SD) rats of similar age were used as control animals. In the insulin-treated group of SDT rats, insulin pellets were implanted at 24 weeks of age to compare the development of renal lesions. The SDT rats began to develop hyperglycemia at 20 weeks of age. In the histopathological examination of the kidney, glycogen deposition of the renal tubular epithelium and renal tubular dilation were observed from 24 weeks of age in the untreated SDT rats, and the changes in the renal tubules markedly progressed with aging. Moreover, thickening of the glomerular basement membrane was observed from 32 weeks of age. At 50 weeks of age, the glomeruli showed increase of mesangial matrix, with predominantly diffuse lesions showing by 68 weeks of age. The mesangial proliferation gradually progressed. In the SD rats, no renal lesions were present at 50 and 68 weeks of age. SDT rats with insulin treatment remained normoglycemic throughout observation and their renal functional parameters were normal. Glycemic control in SDT rats prevented the development of renal lesions. The features of SDT rats indicate their usefulness as an animal model for investigating diabetic nephropathy.     

Apoptosis-Promoting Effects of Hematoporphyrin Monomethyl Ether-Sonodynamic Therapy (HMME-SDT) on Endometrial Cancer

Objective

The aim of the present study was to examine the apoptosis-promoting effects and mechanisms of hematoporphyrin monomethyl ether (HMME)-sonodynamic therapy (SDT) on endometrial cancer cells in vitro.

Methods

Endometrial cancer cell samples were divided into four groups: 1) untreated control group, 2) HMME group, 3) pure ultrasound group, and 4) HMME combined with ultrasound, i.e. SDT group. CCK-8 method was utilized to assess the inhibiting effect of SDT on the proliferation of endometrial cancer cells. Optical microscope and field emission transmission electron microscopy were used to characterize the morphology changes of the cancer cells induced by the treatments. Apoptosis rate, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were examined by flow cytometer. Fluorescence intensity measured by laser scanning confocal microscopy was used to explore the variation of intracellular calcium ion (Ca²⁺) concentration. Apoptosis-related proteins involved in both intrinsic and extrinsic apoptosis signallings were analyzed by western blot.

Results

SDT can effectively induce the apoptosis of endometrial cancer cells. Compared with ultrasound which is known as an effective anti-tumor method, SDT leads to a significant improvement on suppression of cell viability and induction of apoptosis, together with more remarkable modifications on the morphology and substructure in both ultrasound sensitive and resistant endometrial cancer cells. Further studies reveals that SDT promotes ROS production, induces loss of MMP and increases intracellular Ca²⁺ concentration more efficiently than HMME or ultrasound alone. SDT groups also show a rather high expression of apoptosis-promoting proteins, including Bax, Fas and Fas-L, and a significant low expression of apoptosis-suspending proteins including Bcl-2 and Survivin. Meanwhile, both cleaved caspse-3 and caspase-8 are dramatically enhanced in SDT groups. Multiple pathways has been proposed in the process, including the intrinsic activation by excessive ROS and overloaded Ca²⁺, silencing survivin gene, and the extrinsic pathway mediated by the death receptor.

Conclusion

Given its considerable effectivity in both ultrasound sensitive and resistant cells, SDT may therefore be a promising therapeutic method for treating endometrial cancers.     

Effectiveness of Short-Distance Translocation and its Effects on Western Rattlesnakes

ABSTRACT Short-distance translocation (SDT) is commonly used to mitigate snake-human interactions, yet little is known about its effectiveness or its effects on behavior and welfare of snakes. Between April 2004 and October 2005, we evaluated SDT as a conservation and management tool by investigating how 500-m SDT affected spatial ecology, body condition, and behavior of western rattlesnakes (Crotalus oreganus) surgically implanted with radiotransmitters in a field study near Osoyoos, British Columbia, Canada. Of 14 rattlesnakes subjected to SDT, 12 (85.7%) returned on ≥1 occasion (range 1–7 times) to the general area they were removed from. Rattlesnakes that underwent SDT showed an increase in total distance moved over an active season compared to non-translocated snakes, but there was no evidence to suggest SDT had an effect on activity range size. There was no evidence to suggest SDT affected body condition, behavior, or mortality rates. Short-distance translocation to high-quality undisturbed habitats was unsuccessful as a long-term solution to snake-human conflict because most translocated snakes returned to conflict areas within a short time (x̄x = 19.9 ± 8.7 days). However, SDT may be an effective short-term tool to manage snake-human conflict in areas where human presence is seasonal or short-lived if careful attention is paid to species-specific biological needs, habitat quality at the release site, and the location of the release site in relation to conflict areas.     

Genetic analysis for diabetes in a new rat model of nonobese type 2 diabetes,Spontaneously Diabetic Torii rat

The Spontaneously Diabetic Torii (SDT) rat has recently been established as a new rat model of nonobese type 2 diabetes. In this study, we characterized diabetic features in SDT rats, and performed quantitative trait locus (QTL) analysis for glucose intolerance using 319 male (BNxSDT)xSDT backcrosses. Male SDT rats exhibited glucose intolerance at 20 weeks, and spontaneously developed diabetes with the incidence of 100% at 38 weeks, and glucose intolerance is well associated with the development of diabetes. The QTL analysis identified three highly significant QTLs (Gisdt1, Gisdt2, and Gisdt3) for glucose intolerance on rat chromosomes 1, 2, and X, respectively. The SDT allele for these QTLs significantly exacerbated glucose intolerance. Furthermore, synergistic interactions among these QTLs were detected. These findings indicate that diabetic features in SDT rats are inherited as polygenic traits and that SDT rats would provide insights into genetics of human type 2 diabetes.     

Sonodynamic effects of hematoporphyrin monomethyl ether on CNE-2 cells detected by atomic force microscopy

Hematoporphyrin monomethyl ether (HMME) has been effectively used to treat solid tumors of some types. However, its application in nasopharyngeal carcinoma has not been studied yet. In this paper, the detailed sonodynamic effects of HMME‐SDT (sonodynamic therapy) on CNE‐2 cells including cell growth inhibition, apoptosis induction, and membrane toxicity were investigated. It was found that HMME alone had less cytotoxicity whereas HMME‐SDT could suppress the cell proliferation in a dose‐dependent manner as detected by MTT assay. The annexin V‐based flow cytometric data indicated that upon SDT, different concentrations of HMME induce distinct types of cell death, apoptosis by low concentration (60 µg/ml) of HMME and necrosis by higher concentration (120 µg/ml). The immunofluorescence of cytoskeleton and nuclei morphology showed that upon HMME‐SDT, the cells became rounding and the cytoskeletal network disappeared, and, the nuclei represented a total fragmented morphology of nuclear bodies. These alternations showed the apoptosis induction by HMME‐SDT. Further AFM study showed that the cell membrane structure and cytoskeleton networks were destroyed, and, the Young's modulus, tip‐cell‐surface adhesion force decreased to 0.22 ± 0.11 Mpa, 35.4 ± 12.8 pN of cells with 120 µg/ml HMME‐SDT from 0.48 ± 0.21 Mpa, 69.6 ± 22.3 pN of native cells, respectively. These membrane changes caused the collapse of mitochondrial transmembrane potential and disturbance of intracellular calcium homeostasis, which was consistent with the results detected by flow cytometry. Therefore, membrane toxicity and cytoskeleton disrupture induced by HMME‐SDT maybe important factors to induce cell apoptosis, and, the disturbance of mitochondrial transmembrane potential and calcium channels might be the apoptosis mechanisms. J. Cell. Biochem. 112: 169–178, 2011. © 2010 Wiley‐Liss, Inc.     

A novel model of obesity-related diabetes: introgression of the Lepr(fa) allele of the Zucker fatty rat into nonobese Spontaneously Diabetic Torii (SDT) rats.

An fa allele of the leptin receptor gene (Lepr(fa)) of the Zucker fatty rat was introduced into the genome of the Spontaneously Diabetic Torii (SDT) rat, an inbred model of nonobese type 2 diabetes mellitus, through the 'Speed congenic method'. The newly established congenic strain of a SDT rat for Lepr(fa) was maintained by intercrossing between fa-heterozygous littermates, and the phenotypes related to obesity and diabetes were investigated till 32 wks of age. SDT fa/fa rats of both sexes exhibited obesity, adiposity and insulin resistance associated with hyperphagia from the loss of leptin action. Interestingly, they developed diabetes from 5 wks of age in males and 8 wks in females with the incidences reaching 100% at 16 wks in males and 73% at 32 wks in females. In contrast, heterozygous (+/fa) and wild-type (+/+) rats developed spontaneous nonobese diabetes in males from approximately 20 wks, but not in females, as with the original SDT rats. These results indicate that the fa gene accelerates the onset of diabetes in SDT rats by making adiposity and/or insulin resistance as potent risk factors for development of their diabetes. The SDT.Lepr(fa) congenic rat strain is expected to be a novel model of obesity-related diabetes and could be a useful tool for studies of the genetic backgrounds of diabetes in response to fa-induced obesity.     

A Discordant-Sibship Test for Disequilibrium and Linkage: No Need for Parental Data

The sibship disequilibrium test (SDT) is designed to detect both linkage in the presence of association and association in the presence of linkage (linkage disequilibrium). The test does not require parental data but requires discordant sibships with at least one affected and one unaffected sibling. The SDT has many desirable properties: it uses all the siblings in the sibship; it remains valid if there are misclassifications of the affectation status; it does not detect spurious associations due to population stratification; asymptotically it has a chi2 distribution under the null hypothesis; and exact P values can be easily computed for a biallelic marker. We show how to extend the SDT to markers with multiple alleles and how to combine families with parents and data from discordant sibships. We discuss the power of the test by presenting sample-size calculations involving a complex disease model, and we present formulas for the asymptotic relative efficiency (which is approximately the ratio of sample sizes) between SDT and the transmission/disequilibrium test (TDT) for special family structures. For sib pairs, we compare the SDT to a test proposed both by Curtis and, independently, by Spielman and Ewens. We show that, for discordant sib pairs, the SDT has good power for testing linkage disequilibrium relative both to Curtis''s tests and to the TDT using trios comprising an affected sib and its parents. With additional sibs, we show that the SDT can be more powerful than the TDT for testing linkage disequilibrium, especially for disease prevalence >.3.     

血卟啉单甲醚介导的声动力疗法对牙龈卟啉单胞菌生物膜中丙二醛含量的影响

目的:探讨血卟啉单甲醚(hematoporphyrin monomethyl ether,HMME)介导的声动力疗法(sonodynamic therapy,SDT)对牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)生物膜中脂质过氧化物丙二醛(malondialdehyde,MDA)含量的影响。方法:羟基磷灰石片培养Pg生物膜厌氧培养3天,将生物膜随机分为4组(对照组、HMME组、超声组、SDT组),分别与无菌生理盐水或HMME进行避光孵育,然后进行声动力处理。采用平板计数法计算细菌存活率,MDA含量使用MDA检测试剂盒在可见光分光光度计下进行检测。结果:当超声强度为3 W/cm~2,超声时间为5 min时,SDT组的生物膜细菌存活率仅为40%,与对照组相比显著降低(P0.05),超声组细菌存活率为62%,与对照组相比亦显著降低(P0.05)。相同超声参数下,SDT组Pg生物膜中MDA含量最高,达17.3±1.2 nmol/mL(P0.05),超声组生物膜中MDA含量为7±0.8 nmol/mL,与对照组相比差异并无统计学意义(P0.05)。结论:HMME介导的SDT对Pg生物膜有一定杀伤效果,并且在杀伤过程中,可引发脂质过氧化反应,导致MDA释放。     

Contribution of hyperglycemia on diabetic complications in obese type 2 diabetic SDT fatty rats: effects of SGLT inhibitor phlorizin

The spontaneously diabetic torii (SDT) fatty rat is a new model of type 2 diabetes showing overt obesity, hyperglycemia and hyperlipidemia. With early onset of diabetes mellitus, diabetic microvascular complications, including nephropathy, peripheral neuropathy and retinopathy, are observed at young ages. In the present study, blood glucose levels of female SDT fatty rats were controlled with phlorizin, a non-selective SGLT inhibitor, to examine whether and how these complications are caused by hyperglycemia. Phlorizin treatment adequately controlled plasma glucose levels during the experiment. At 29 weeks of age, urinary albumin excretion considerably increased in SDT fatty rats. Glomerulosclerosis and tubular pathological findings also indicate diabetic nephropathy. These renal parameters tended to decrease with phlorizin; however, effects were partial. Sciatic nerve conduction velocities were significantly delayed in SDT fatty rats compared with Sprague-Dawley (SD) rats. Intraepidermal nerve fiber density, an indicator of subclinical small nerve fiber neuropathy, significantly decreased in SDT fatty rats. Retinal dysfunction (prolongation of peak latency for oscillatory potential in electroretinograms) and histopathological eye abnormalities, including retinal folding and mature cataracts were also observed. Both nerve and eye disorders were prevented with phlorizin. These findings indicate that severe hyperglycemia mainly causes diabetic complications in SDT fatty rats. However, other factors, such as hyperlipidemia and hypertension, may affect diabetic nephropathy. These characteristics of diabetic complications will become helpful in evaluating new drugs for diabetic complications using SDT fatty rats.     

Establishment of an Animal Model of Bisphosphonate-Related Osteonecrosis of the Jaws in Spontaneously Diabetic Torii Rats

Background

We evaluated the side effects of bisphosphonate (BP) on tooth extraction socket healing in spontaneously diabetic Torii (SDT) rats, an established model of non-obese type 2 diabetes mellitus, to develop an animal model of BP-related osteonecrosis of the jaws (BRONJ).

Materials and Methods

Male Sprague-Dawley (SD) rats and SDT rats were randomly assigned to the zoledronic acid (ZOL)-treated groups (SD/ZOL or SDT/ZOL) or to the control groups (SD/control or SDT/control). Rats in the SD/ZOL or SDT/ZOL groups received an intravenous bolus injection of ZOL (35 μg/kg) every 2 weeks. Each group consisted of 6 rats each. Twenty-one weeks after ZOL treatment began, the left maxillary molars were extracted. The rats were euthanized at 2, 4, or 8 weeks after tooth extraction, and the total maxillae were harvested for histological and histochemical studies.

Results

In the oral cavity, bone exposure persisted at the tooth extraction site in all rats of the SDT/ZOL group until 8 weeks after tooth extraction. In contrast, there was no bone exposure in SD/control or SDT/control groups, and only 1 of 6 rats in the SD/ZOL group showed bone exposure. Histologically, necrotic bone areas with empty lacunae, microbial colonies, and less invasion by inflammatory cells were observed. The number of tartrate-resistant acid phosphatase-positive osteoclasts was lower in the SDT/ZOL group than in the SD/control group. The mineral apposition rate was significantly lower in the SDT/ZOL group compared with the SD/control group.

Conclusions

This study demonstrated the development of BRONJ-like lesions in rats and suggested that low bone turnover with less inflammatory cell infiltration plays an important role in the development of BRONJ.     

An empirical test of signal detection theory as it applies to Batesian mimicry

Signal detection theory (SDT) has been repeatedly invoked to understand how palatable prey might gain an advantage by resembling unpalatable prey. Here we developed an experimental test of the theory in which we sequentially presented computer-generated Mimics (profitable to attack) and Models (unprofitable to attack) to human volunteers, and asked them to forage in a way that maximized their personal scores. Both the Mimics and Models exhibited normally distributed variation in a single stimulus dimension. When we varied the mean similarity of Mimics to Models, and the proportion of all prey items that were Mimics, our human predators made foraging decisions that were close to those predicted by SDT, including the adoption of a threshold in appearance beyond which prey items were unlikely to be attacked. The fit of predictions to observations was marginally closer when including the time taken to handle the two types of prey. When Mimics and Models were allowed to evolve in appearance subject to selection, the evolutionary trajectory fitted the predictions of SDT closely. While our system was not appropriate to test all predictions of recent SDT theory, it provides strong support for the SDT framework as it applies to Batesian mimicry.     

Heart angiotensin II-induced cardiomyocyte hypertrophy suppresses coronary angiogenesis and progresses diabetic cardiomyopathy

To examine whether and how heart ANG II influences the coordination between cardiomyocyte hypertrophy and coronary angiogenesis and contributes to the pathogenesis of diabetic cardiomyopathy, we used Spontaneously Diabetic Torii (SDT) rats treated without and with olmesartan medoxomil (an ANG II receptor blocker). In SDT rats, left ventricular (LV) ANG II, but not circulating ANG II, increased at 8 and 16 wk after diabetes onset. SDT rats developed LV hypertrophy and diastolic dysfunction at 8 wk, followed by LV systolic dysfunction at 16 wk, without hypertension. The SDT rat LV exhibited cardiomyocyte hypertrophy and increased hypoxia-inducible factor-1α expression at 8 wk and to a greater degree at 16 wk and interstitial fibrosis at 16 wk only. In SDT rats, coronary angiogenesis increased with enhanced capillary proliferation and upregulation of the angiogenic factor VEGF at 8 wk but decreased VEGF with enhanced capillary apoptosis and suppressed capillary proliferation despite the upregulation of VEGF at 16 wk. In SDT rats, the phosphorylation of VEGF receptor-2 increased at 8 wk alone, whereas the expression of the antiangiogenic factor thrombospondin-1 increased at 16 wk alone. All these events, except for hyperglycemia or blood pressure, were reversed by olmesartan medoxomil. These results suggest that LV ANG II in SDT rats at 8 and 16 wk induces cardiomyocyte hypertrophy without affecting hyperglycemia or blood pressure, which promotes and suppresses coronary angiogenesis, respectively, via VEGF and thrombospondin-1 produced from hypertrophied cardiomyocytes under chronic hypoxia. Thrombospondin-1 may play an important role in the progression of diabetic cardiomyopathy in this model.     

A New Spontaneously Diabetic Non-obese Torii Rat Strain With Severe Ocular Complications

A new spontaneously diabetic strain of the Sprague-Dawley rat was established in 1997 and named the SDT (Spontaneously Diabetic Torii) rat. In this research, we investigated the characteristics of the disease condition in the SDT rats. The time of onset of glucosuria was different between male and female SDT rats; glucosuria appeared at approximately 20 weeks of age in male rats and at approximately 45 weeks of age in female rats. A cumulative incidence of diabetes of 100% was noted by 40 weeks of age in male rats, while it was only 33.3% even by 65 weeks of age in female rats. The survival rate up to 65 weeks of age was 92.9% in male rats and 97.4% in female rats. Glucose intolerance was observed in male rats from 16 weeks of age. The clinical characteristics of the male SDT rats were (1) hyperglycemia and hypoinsulinemia (from 25 weeks of age); (2) long-term survival without insulin treatment; (3) hypertriglyceridemia (by 35 weeks of age); however, no obesity was noted in any of the male rats. The histopathological characteristics of the male rats with diabetes mellitus (DM) were (1) fibrosis of the pancreatic islets (by 25 weeks of age); (2) cataract (by 40 weeks of age); (3) tractional retinal detachment with fibrous proliferation (by 70 weeks of age) and (4) massive hemorrhaging in the anterior chamber (by 77 weeks of age). These clinical and histopathological characteristics of the disease in SDT rats resemble those of human Type 2 diabetes with insulin hyposecretion. In conclusion, SDT rat is considered to be a potentially useful model for studies of diabetic retinopathy encountered in humans.     

Effect of different warm-up procedures on subsequent swim and overall sprint distance triathlon performance

This study investigated the effect of 3 warm-up procedures on subsequent swimming and overall triathlon performance. Seven moderately trained, amateur triathletes completed 4 separate testing sessions comprising 1 swimming time trial (STT) and 3 sprint distance triathlons (SDT). Before each SDT, the athletes completed 1 of three 10-minute warm-up protocols including (a) a swim-only warm-up (SWU), (b) a run-swim warm-up (RSWU), and (c) a control trial of no warm-up (NWU). Each subsequent SDT included a 750-m swim, a 500-kJ (～20 km) ergometer cycle and a 5-km treadmill run, which the athletes performed at their perceived race intensity. Blood lactate, ratings of perceived exertion, core temperature, and heart rate were recorded over the course of each SDT, along with the measurement of swim speed, swim stroke rate, and swim stroke length. There were no significant differences in individual discipline split times or overall triathlon times between the NWU, SWU, and RSWU trials (p > 0.05). Furthermore, no difference existed between trials for any of the swimming variables measured (p > 0.05) nor did they significantly differ from the preliminary STT (p > 0.05). The findings of this study suggest that warming up before an SDT provides no additional benefit to subsequent swimming or overall triathlon performance.