Numerous chemical compounds are distributed around the world and may affect the homeostasis of the endocrine system by disrupting the normal functions of hormone receptors. Although the risks associated with these compounds have been evaluated by acute toxicity testing in mammalian models, the chronic toxicity of many chemicals remains due to high cost of the compounds and the testing, etc. However, computational approaches may be promising alternatives and reduce these evaluations. Recently, deep learning (DL) has been shown to be promising prediction models with high accuracy for recognition of images, speech, signals, and videos since it greatly benefits from large datasets. Recently, a novel DL-based technique called DeepSnap was developed to conduct QSAR analysis using three-dimensional images of chemical structures. It can be used to predict the potential toxicity of many different chemicals to various receptors without extraction of descriptors. DeepSnap has been shown to have a very high capacity in tests using Tox21 quantitative qHTP datasets. Numerous parameters must be adjusted to use the DeepSnap method but they have not been optimized. In this study, the effects of these parameters on the performance of the DL prediction model were evaluated in terms of the loss in validation as an indicator for evaluating the performance of the DL using the toxicity information in the Tox21 qHTP database. The relations of the parameters of DeepSnap such as (1) number of molecules per SDF split into (2) zoom factor percentage, (3) atom size for van der waals percentage, (4) bond radius, (5) minimum bond distance, and (6) bond tolerance, with the validation loss following quadratic function curves, which suggests that optimal thresholds exist to attain the best performance with these prediction models. Using the parameter values set with the best performance, the prediction model of chemical compounds for CAR agonist was built using 64 images, at 105° angle, with AUC of 0.791. Thus, based on these parameters, the proposed DeepSnap-DL approach will be highly reliable and beneficial to establish models to assess the risk associated with various chemicals. However, the risk remains that such compounds could pose major public health concerns to humans by potentially disrupting normal endocrine functions with various hormone receptors upon long-term exposure  that constitutes DL has a function of feature expression learning that makes it automatically extract features and unnecessary to manually extract features  in the United States  program, a multi-agency collaboration between the U.S. Environmental Protection Agency, the National Institute of Environmental Health Sciences, National Toxicology Program, NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, and the US Food and Drug Administration (1) incorporate advances in molecular systems by identifying patterns of chemical compounds-induced biological response, (2) prioritize compounds for more extensive toxicological evaluation, and (3) develop predictive models for biological response in human NRC., . Their shttps://www.mn-am.com/products/corina) has been licensed in the past to predict 3D structures for some of the molecules in the main large public databases of small molecules such as PubChem a data-based commercial 3D molecular model builder with high accuracy and high speed for the 3D-structures of organic and metal-organic  molecules high coverage for nearly all organics but approximately half of the organometallics  format −1 or f (x) = tanh (x) because the training time with gradient descent ReLUs much faster than that associated with if the input is negative, there is no contribution to other units  images contained digitized information data about plane configuration and the corresponded to the type of atom for the chemical structure produced by Deep Snap were resized by DIGITS version 4.0.0 software to a fixed resolution of 256 × 256 pixels and input into DL model to build the prediction models, which were trained based on the activity scores of chemical compounds and the corresponding 2D chemical-structure images. In this study, the total number of training epochs was 30, snapshot interval in epochs 1, validation interval in epochs 1, random seed 1, solver type stochastic gradient descent, base learning rate 0.01. Training, testing, and validation were performed using the dataset described in i, respectively.The loss, which is a summation (not a percentage) of the errors in each dataset as shown below cross entropy error (CEE) with respect to the model's parameters by changing the weight vector values, in construction of the prediction models is calculated on training and validation datasets, where pi and yi correspond to the accuracy label (ground truth vector) and output of softmax  for class The loss value implies how well or poorly a certain model behaves after each iteration of optimization. Loss is indicative of unless the model has over-fitted with respect to the training data. The accuracy of the model is usually determined after the validation samples are fed to the model and the number of mistakes (zero-one loss) that the model makes recorded. The percentage of misclassification is calculated  labels, and the auroc (area under receiver operating characteristic) was calculated using JMP pro 14, statistical discovery software (SAS Institute Inc. NC) to evaluate the predictive models using 3D chemical structures including training , validation , and testing  datasets captured from eight increments on the x-, y-, and z-axes: , , , , , , , and   Linden,. SensitiSpecificity is the true negative rate i.e., the proportion of actual negative samples that were correctly identified as negative for all negative samples including true and false negatives.The file, including chemical structures as indicated by SMILES, chemical annotation numbers, activity scores, dataset classes divided into training and validation. Based on this information, the 3D chemical structures were built, descriptors were calculated using the MOE chemical calculation system. Using these descriptors, the prediction model was constructed using JMP pro 14.2 values) of the AUC with each epoch were 0.95  were calculated from the testing datasets with validation loss (VL), training loss (TL), and validation accuracy (VA). VL is an error summation not a percentage obtained from how well the model is doing for. TL is an error summation which by attempting to determine good values for all the weights and biases . VA is the percentage of correct answers based on the results obtained from. As results, these R2 values with AUCs were 0.86 (VL), 0.62 (TL), and 0.57 (VA), respectively  and validation  datasets 180° angle . The tesere 0.95 . The corectively . Moreoveectively . These f2 values between these parameters and VLs were more than 0.90, and each of these relations followed quadratic function curves. Also, the R2 values of the running time (RT) in DL with the above six parameters showed that the RTs were moderately associated with AT (R2 = 0.48), BR (R2 = 0.47), and BT (R2 = 0.43) , , , and  in the training , validation , and test  datasets . The fol = 0.43) . However = 0.43) . Similar = 0.43) . The IPS = 0.43)  and mini = 0.43) , respect = 0.43) . Also, w = 0.43)  on the o = 0.43) , left. H = 0.43) , right.  = 0.43) , right.  = 0.43) , right.  = 0.43) , left. Thttps://pubchem.ncbi.nlm.nih.gov/#, AID 1224892). Therefore, it is necessary to optimize various types of assignments for the activity scores and/or other datasets in detail to further increase the prediction performance. In addition, a comparison of the performances between this state-of-the-art Deep Snap and 1,024 of extended-connectivity fingerprint (ECFG) of descriptors calculated from Dragon 7.0  the suitableness of input as supervised data, (2) sufficient amount of images for training, and (3) adequate training for input dataset of pictures of chemical structure into the DL, the activity scores of the datasets, including training, validation, and test, were randomly assigned keeping the numbers of the three datasets unchanged as permutation test. The calculation of the performed each parameterized values of Deep Snap with each best performance model to capture chemical structures eight pictures at 180° angle using a total of ten different datasets with assignments of various activity scores. As result, the average AUCs were 0.553 (±0.007) with the average minimum VL of 0.522 (±0.014), indicated almost random guessing. These results suggest that the prediction models in this study extracted the CAR agonist activity-specific structural features from chemical compounds. Also, we calculated the AUC random forest as another method the same datasets for the above Deep Snap for CAR agonist and 206 of descriptors to build the prediction model in ROC-AUC value 0.749. Previously, we found that the prediction for the performance of compounds inducing MMP disruption was better 45° angles using 512 pictures for one molecule, with AUCs of 0.921 Uesawa, . MoreoveWhen considering applying DL to a compound, whose molecular structure is a variable data format that can have branches and loops, there are problems with how to handle that input or output. To address this issue, graphic-based convolution, which has the ability to handle graph structures, simple encoding of the molecules  represented by edge-connected nodes introducing convolution operations on each nodes non-Euclidean structure was proposed as modifications of DL architectures specialized for molecular fingerprints and models in the terms of structural features, physical properties, and activity  extract spatiotemporal features from moving objects proposed as a method applied to motion image recognition  number of molecules per SDF files split into (2) zoom factor percentage, (3) atom size for van der waals percentage, (4) bond radius, (5) minimum bond distance, and (6) bond tolerance with the VLs as indicator for evaluating the performance of the DL following quadratic function curves, suggesting that optimal thresholds exist to attain the best performance with these prediction models. Using the parameter values the best performance with the prediction model, the prediction model for CAR agonist was built using 64 images at 105° angle AUCs of 0.791. The results of this study feature the possible power of novel DL-based QSAR approach for prediction of potential toxicity of large datasets of any chemical compounds.YU initiated and supervised the work, designed the experiments, collected the information about chemical compounds, and edited the manuscript. YM drafted the manuscript. YU and YM read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Diabetes is a complex metabolic disease characterized by chronic low-grade inflammation in which genetic and environmental factors are involved. Growing evidence implicates that alterations of the gut microbiota potentially contribute to the emergence of metabolic diseases. The human appendix has more recently been recognized as a microbial reservoir for repopulating the gastrointestinal tract and an important part of the immune system. Thus, appendectomy may influence microbial ecology and immune function. This study investigated the association between appendectomy and type 2 diabetes risk.We analyzed a cohort of 10954 patients who underwent appendectomy between 1998 and 2013 based on the Taiwan National Health Insurance Program database. A comparison cohort of 43815 persons without appendectomy was selected randomly and matched by sex, age, comorbidities, and index year. To ensure reliability of the results, a sensitivity analysis using a propensity score–matched study was performed. We observed the subsequent development of type 2 diabetes in both cohorts.interaction = 0.002); in contrast, sex did not affect the association between appendectomy and type 2 diabetes risk (Pinteraction = 0.88).Although the overall incidence of type 2 diabetes in the appendectomy patients was 7.9% higher than that in the non-appendectomy patients, it was not statistically significant  after the adjustment of confounding factors. Multivariate regression analysis revealed that the adjusted hazard ratio (HR) of type 2 diabetes was 1.347 for appendectomy patients < 30 years of age  compared to non-appendectomy patients. The incidence of type 2 diabetes was higher within 3 years of post-appendectomy follow-up than for non-appendectomy patients . Age impacted the association between appendectomy and type 2 diabetes risk (POur study results suggest that appendectomy increases type 2 diabetes risk, particularly when performed prior to middle age. Diabetes is one of the most serious public health issues worldwide due to its rapidly increasing incidence and tremendous amount of various vascular complications. According to the International Diabetes Federation, in 2015, the 415 million individuals had diabetes, a figure that is estimated to increase to 642 million by 2040  that invDespite long being considered an evolutionary vestigial organ, the human appendix has more recently been recognized as an important part of the immune system. Furthermore, the human appendix may function as a “safe house” to preserve and protect commensal bacteria in the gastrointestinal tract via immune-mediated biofilm formation –15. ConsInformed consent statement. Although written informed consent was not provided by the participants for the use of their medical records in this study, the patient records were de-identified and anonymized prior to the investigation. The work was approved after a full ethical review by the Institutional Review Board (IRB) of the Changhua Christian Hospital , and the IRB waived the need for consent.th Revision, ICD-9 procedure codes 47.0 and 47.1). We identified a study group of 15917 patients who underwent appendectomy between 1998 and 2013. We excluded 2975 persons aged <18 or >100 years, 1538 persons with a history of DM history before the index date, 201 patients with any antidiabetic agent history before the index date, eight patients who underwent bariatric surgery, and 241 patients who did not survive or were followed up for < 30 days. The remaining 10954 patients were included in the study as the appendectomy group. The index year was determined as the year the appendectomy was performed. We established a random frequency-matched study cohort for assessing the association between appendectomy and DM. For each identified appendectomy patient, four control groups were frequency matched by age, sex, and index year. A total of 10954 appendectomy patients and 43815 controls were included in the study cohort . Started in 1995, the program includes 99% of the 23-million-plus people who live in Taiwan. This database from the NHIP is managed and established for investigators. With the hospitalization database from the Taiwan NIHP, the appendectomy group was determined as patients aged 20–84 years undergoing appendectomy from 1998 to 2013 , hyperlipidemia (ICD-9 272), gout (ICD-9 274), polycystic ovaries (ICD-9 256.4), cerebrovascular disease (ICD-9 430–438), renal disease (ICD-9 580–589), ischemic heart disease , chronic obstructive pulmonary disease , gestational diabetes (GDM) , depression , obesity , chronic pancreatitis (ICD-9 577.1), hepatitis B infection , and hepatitis C infection .The major outcome was a new diagnosis of DM (ICD-9 250.0–250.9) based on at least three outpatient diagnoses or one hospital discharge diagnosis during the follow-up period. All study subjects were followed until they were diagnosed with DM at the end of 2013.Demographic and clinical characteristics in the appendectomy and non-appendectomy subjects were presented as proportions and mean ± standard deviation (SD). The standardized differences were used with an absolute value for the standardized difference of >0.1 considered to indicate important imbalances between the two groups for categorical or continuous matching variables, respectively. The incidence of DM was measured as the event number of DM diagnosed during the follow-up year divided by the total follow-up person-years for each subject.The Cox proportional hazards regression model was applied to measure the hazard ratio (HR) and 95% confidence interval (CI) for the risk of DM in the appendectomy compared to non-appendectomy groups. Multivariate Cox analysis with cause-specific hazard models was used to estimate adjusted hazard ratios (aHRs) adjusted for confounders for appendectomy and other comorbidities including hypertension, hyperlipidemia, gout, polycystic ovaries, depression, cerebrovascular disease, renal disease, CAD, COPD, GDM, obesity, chronic pancreatitis, HBV infection, HCV infection, medications  and number of visiting clinics.Uni- and multivariate survival analyses were performed for the incidence and relative risk of DM. A subgroup analysis was used to distinguish between DM risks in the appendectomy and non-appendectomy groups. We then used propensity score-matched analysis to assess the reliability of our results. Propensity scores were calculated using multivariate logistic regression to predict the probability of DM occurrence. The cumulative incidence curves from cause-specific hazard methods for the appendectomy and control groups were also analyzed.Of the 15917 patients in the appendectomy group, 10954 patients remained after the exclusion of 2975 patients <18 or >100 years of age, 1538 patients with a history of DM, 201 patients with a prescription history of antidiabetic agents, eight patients who underwent bariatric surgery, and 241 patients who did not survive or completed <30 days of follow-up. There were a total of 43815 persons in the control group.Comparisons in demographic characteristics are shown in interaction = 0.002), whereas sex did not (Pinteraction = 0.88).To assess the reliability of cohort 1, a propensity score-matched sensitivity analysis (cohort 2) was conducted, and it showed that the appendectomy and control groups had equal distributions of all demographic characteristics. Overall, 7.4% (n = 811) of patients with appendectomy developed type 2 DM versus 6.91% (n = 3027) of persons without appendectomy in cohort 1. Similar results were shown in cohort 2. Using this data mining strategy, we were able to examine the correlation between appendectomy and the development of type 2 DM. As shown in The incidence of type 2 DM stratified by age is shown in Moreover, the cumulative incidences are shown in In this large retrospective population-based cohort study, we found no statistically significant association between appendectomy and type 2 DM in the overall population . However, appendectomy increased the risk of type 2 DM among patients < 20 years  and those <30 years . In the random frequency-matched cohort, all confounding factors except Charlson Comorbidity Index score and number of visiting clinics were equally distributed in the appendectomy and control groups. To reduce selection bias, a multivariate regression analysis was conducted of a propensity score-matched cohort, which revealed that the adjusted HR of type 2 DM in the appendectomy patients < 20 years and those < 30 years were 2.52  and 1.36  compared to controls. Since the number of appendectomy patients < 20 years of age was limited, we may have overestimated the impact of age on appendectomy and the development of type 2 DM. Therefore, we concluded that appendectomy prior to 30 years of age is an independent risk factor for the development of type 2 DM.interaction = 0.88). The aHR of type 2 DM incidence in the male appendectomy group was 1.058 (P = 0.34), while that in the female appendectomy group was 1.072 (P = 0.20) compared to controls. Furthermore, the major outcome was a new diagnosis of DM (ICD-9 250.0–250.9), which included type 1 and 2 DM. Because patients with a history of DM before the index year and subjects < 18 years were excluded, we believe that most of newly diagnosed DM patients after appendectomy have type 2 DM.Apart from significant results in the age subgroup analyses confirmed by the different statistical methods, our results were adjusted by many confounding factors, including hypertension, hyperlipidemia, gout, polycystic ovaries, depression, GDM, depression, obesity, chronic pancreatitis, HBV, HCV, statins, atypical antipsychotics, HIV drugs, corticosteroids, and immunosuppressants. This implies that age indeed had a profound effect on the association between appendectomy and type 2 DM. On the other hand, we found that sex did not influence the association between appendectomy and type 2 DM Click here for additional data file.


CLDN14, was reported to be responsible for human hereditary hearing loss, DFNB29. Until now, nine pathogenic variants have been reported, and most phenotypic features remain unclear. In the present study, genetic screening for 68 previously reported deafness causative genes was carried out to identify CLDN14 variants in a large series of Japanese hearing loss patients, and to clarify the prevalence and clinical characteristics of DFNB29 in the Japanese population. One patient had a homozygous novel variant (c.241C>T: p.Arg81Cys) (0.04%: 1/2549). The patient showed progressive bilateral hearing loss, with post-lingual onset. Pure-tone audiograms indicated a high-frequency hearing loss type, and the deterioration gradually spread to other frequencies. The patient showed normal vestibular function. Cochlear implantation improved the patient’s sound field threshold levels, but not speech discrimination scores. This report indicated that claudin-14 is essential for maintaining the inner ear environment and suggested the possible phenotypic expansion of DFNB29. This is the first report of a patient with a tight junction variant receiving a cochlear implantation.Tight junctions are cellular junctions that play a major role in the epithelial barrier function. In the inner ear, claudins, occludin, tricellulin, and angulins form the bicellular or tricellular binding of membrane proteins. In these, one type of claudin gene,  CLDN14 gene.Hearing loss (HL) is the most common sensory impairment and is diagnosed in approximately two in every 1000 children . The inh+ and low Na+ concentrations [+ concentration and EP) are indispensable for cochlear hair cells to transduce acoustic stimuli into electrical signals [LSR, angulin-2/ILDR1, and angulin-3/ILDR2) form the bicellular or tricellular binding of membrane proteins. Among these, claudin-14, tricellulin, and angulin-2 are reported to be responsible for human deafness DFNB29, DFNB49, and DFNB42, respectively [For our sound receiving process, it is necessary to convert sound vibration to nerve action potential (mechano-transduction) . The centrations . Furthertrations ,10. It i signals . The bar signals . Tight jectively ,14,15.Cldn9 mutant mice and Cldn14 mutant mice demonstrate hearing loss. However, these two animal models do not display reduced EP [The claudins are a family of proteins that play a major role in epithelial barrier function, especially in bicellular junctions. There are 24 claudins that have been identified in humans thus far ,16,17,18duced EP ,23. The CLDN14 [CLDN14 variants are a relatively common cause of recessive hearing loss, which is responsible for 2.25% of HL patients in a Pakistani study cohort [CLDN14 variants, the audiovestibular findings, such as progression and details of vestibular function, remain unclear. In addition, there are no reports on patients with cochlear implantation (CI), thus the outcome of CI is also unknown. In the present study, we used massively parallel DNA sequencing (MPS) to detect pathogenic variants in CLDN14 among a large series of Japanese HL patients. To date, nine different variants in CLDN14 ,27,28,29y cohort ,31. AlthCLDN14 gene identified from a group of non-consanguineous HL patients and discuss additional clinical features including CI outcomes. This is the first report of a diagnosis of hearing caused by CLDN14 in an east Asian population and in such a patient receiving a cochlear implant as intervention.Here, we present a patient with a novel variant in the CLDN14: NM_144492: c.241C>T, leading to p.Arg81Cys, was homozygously detected in one of 1577 probands with autosomal recessive HL , and almost all scores indicated “damaging” . The corThe proband was a 37-year-old female. The newborn hearing screening program had not yet started at birth and there were no particular complications in the perinatal period. The female proband also passed an elementary school health checkup at six years of age, but was suspected of hearing loss at age nine and was referred to an otolaryngology clinic at a local general hospital. Pure-tone audiograms showed steep high-frequency sensorineural HL (SNHL) with an average of 77 dBHL in both ears A. It appThe proband underwent a CI (MED-EL FLEX28) in the right ear at the age 35. Sound field threshold levels were improved from 77.5 dBSPL (with hearing aids) to 37.5 dBSPL (with the cochlear implant) A. To evaCLDN14 gene as a cause of progressive SNHL in a Japanese patient. To date, a total of nine pathogenic variants in CLDN14 have been reported  among the Japanese HL patients, and 0.06% (1/1577) among the families with autosomal recessive HL in the Japanese population. In clear contrast to this, variants of CLDN14 account for 2.25% of autosomal recessive HL in the Pakistani population. In this study, the patient was identified from a non-consanguineous family and the parents of the proband were from a geographically remote area, whereas almost all of the previous reports were from consanguineous families [CLDN14 variants were found in our cohort, but one HL patient with a homozygous CLDN14 variant was identified, it is necessary to consider such rare HL cases.The incidence of families ,27,28,29CLDN14 remains unclear, and further study including serial audiograms is required to clarify this. Until Pater et al. reported the progression of HL , the pheCldn14 knockout model mice has not been reported and the details remain unknown. This is the first report to mention in detail the vestibular function, including caloric testing, cVEMPs, and oVEMPs. Endolymph in cochlear is characterized by extremely high resting potential called EP, in addition to high K+ concentration. In contrast, in the vestibular endolymph, the K+ concentration is high, but no EP can be detected. Claudin-14 may be necessary for maintaining a barrier to resist EP, but not K+.We could not identify any dysfunction on the vestibular examinations. According to past reports, claudin-14 is also expressed in the vestibule of mice (such as in the sensory epithelia) . VestibuCLDN14-associated HL patient. A CI represents the most successful neural prosthesis in clinical cases [This is the first report showing the outcome for a CI for a al cases . For patal cases . The impal cases .CLDN14 was expressed at the same level in the organ of Corti and spiral ganglions [As mentioned above, the patient showed relatively poor improvement in speech discrimination scores, whereas good improvement in sound field threshold levels was observed. Since the cochlear implant directly stimulates the cochlear nerve, the influence of the inner ear should be excluded. Thus, it is likely that the unsatisfactory speech discrimination was due to deficiencies in the spiral ganglion or central auditory pathway. In our previous study, anglions . IncludiAll procedures were approved by the Shinshu University Ethical Committee (No. 387—4 September 2012 and No. 576—2 May 2017) as well as the respective Ethical Committees of the other participating institutions described elsewhere  and wereFor the genetic analysis for this proband, we performed target genome enrichment for 68 previously reported genetic causes for deafness and MPS analysis described elsewhere . In brieThe missense, nonsense, insertion/deletion, and splicing variants were selected from among the identified variants. Variants were further selected as less than 1% of (1) the ExAC ,43, (2) CLDN14 variant.The age of onset of HL, the incidence of progressive HL, and episodes of vertigo and dizziness were analyzed based on the medical charts of the probands with the Pure-tone audiometry was performed to evaluate HL. The pure-tone average (PTA) was calculated from the audiometric thresholds at four frequencies . If an individual did not respond to the maximum hearing level at a frequency, 5 dB was added to the maximum hearing level. The severity of HL was classified into mild (PTA: 20–40 dBHL), moderate (41–70 dBHL), severe (71–95 dBHL), and profound (>95 dBHL). The audiometric configurations were categorized as low-frequency, mid-frequency, high-frequency (gently or steeply sloping), flat, and deaf . The vestibular examination findings, including caloric testing and the measurement of cervical and ocular vestibular evoked myogenic potentials (c/oVEMPs), were analyzed. Caloric testing involved the measurement of the maximum slow phase velocity (SPV) by cold water irrigation . We defined a maximum SPV value below 10 deg/s as representing areflexia and a value between 10 and 20 deg/s as representing hyporeflexia. For cVEMPs testing, electromyography (EMG) was performed using a pair of surface electrodes mounted on the upper half and sternal head of the sternocleidomastoid muscle, respectively. The electrographic signal was recorded using a Neuropack evoked potential recorder . Clicks lasting for 0.1 ms at 105 dBnHL were presented through a headphone. The stimulation rate was 5 Hz, the bandpass filter intensity was 20 to 2000 Hz, and the analysis time was 50 ms. The responses to 100 stimuli were averaged twice. The oVEMPs testing was measured by bone-conductive vibration (BCV). The BCV was delivered in 4 ms tone bursts of 500 Hz vibration  by using a hand-held 4810 mini-shaker , which was placed on the midline (Fz) of the forehead. The active electrode was located over the inferior orbital margin and a reference electrode was placed 2 cm below the active electrode. The ground electrode was placed on the chin. The patients laid in a supine position on a bed and looked up at an angle of approximately 30 degrees above straight ahead during the recording. The signals were amplified and bandpass filtered between 20 and 2000 Hz. The stimulus intensity was 115 dB force level, 500 Hz with an analysis time of 40 ms, and 50 responses were averaged for each run. The VEMPs asymmetry was calculated as follows: asymmetry ratio (AR) =  × 100/. In this study, an asymmetry ratio of >30% was defined as a decreased reaction and no reaction in amplitude VEMPs as absent.Intervention for HL, including the use of hearing aids or cochlear implants, was investigated. To evaluate speech perception outcomes, speech discrimination scores (using the 67S Japanese monosyllable test) were used. The subjects sat 1 m away from the sound source facing zero-degree azimuth, and recorded monosyllable words in quiet were presented in the sound field at 65 dBSPL.CLDN14 gene identified from a non-consanguineous family. This is the first report of CLDN14-associated HL in an east Asian population. Serial audiograms indicated high-frequency hearing loss type, and the deterioration gradually spread to other frequencies, finally resulting in deafness. The patient showed normal vestibular function for caloric testing, cVEMPs, and oVEMPs. This is also the first report of an HL patient with a tight junction variant receiving a CI. The CI improved the proband’s sound field threshold levels, but not their speech discrimination scores. This information contributes to our understanding, diagnosis, and treatment of HL caused by TJ disorders.We present a patient with a novel variant in the


Amifostine is well known cytoprotector which is efficient when administered before a wide range of antineoplastic agents. The aim of our study was to investigate amifostine effects on doxorubicin-induced toxic changes in rats. Amifostine (75 mg/kg ip) was given 30 min before each dose of doxorubicin . The animals’ whole-body, liver, and kidney weight, serum biochemical examination, as well as microscopic examination of bone marrow, peripheral blood, liver, and kidney, were done on day 56 of the study. Hepatic and renal alterations were carefully quantified by semiquantitative grading scales—hepatic and renal damage score, respectively. In amifostine-pretreated rats, the number of peripheral blood leukocytes was significantly higher in comparison to doxorubicin-only treated group, preferentially protecting neutrophils. In the same group of rats, hepatic and renal alterations associated with polymorphonuclear cell infiltrates were significantly less severe than those observed in animals receiving only doxorubicin. Our results showed that amifostine successfully protected rats against multiple-dose doxorubicin-induced toxicity by complex, and still not fully elucidated mechanisms of action. Doxorubicin (DOX) is an anthracycline antibiotic that is used as an antineoplastic agent in hematological, as well as in solid malignancies, due to its high antitumor efficacy ,4,5,6,7.It is well known that DOX is able to interfere with a number of biochemical functions within cells, but the precise molecular pathogenesis of both therapeutic and toxic effects are still controversial ,5. There2−), hydroxyl radicals (OH·), and hydrogen peroxide, which induce lipid peroxidation and protein oxidation [As far as the liver is concerned, DOX is metabolized predominantly by liver microsomal enzymes and cytoplasmic reductase ,21 to thxidation ,24, and xidation . Since hxidation . It is axidation ,19,26. Exidation ,27. Furtxidation ,28.It is considered that the successful prevention or treatment of these DOX-induced harmful effects depends on antioxidative potential of different cytoprotective agents and their ability to react with highly reactive oxygen species (ROS) ,30,31,32Amifostine (AMI) is a thiophosphate derivative of cysteamine, which, if administered before cytotoxic chemotherapy, provides protection of various normal tissue, without attenuating their antitumor response ,34,35,36Although DOX toxic effects on the liver and kidney are probably mediated initially by highly reactive free radical formation, we hypothesized that final irreversible changes are the result of inflammatory reactions involving cells of bone marrow origin, like was previously demonstrated in the rat model of chronic cardiac toxicity induced by DOX .Therefore, our aim was to investigate AMI effects on DOX-induced toxic changes in bone marrow, liver and kidney, and their contribution to the significantly better survival of the rats treated by multiple, low doses of DOX.Visible signs of general weakness, with hemorrhagic diarrhea, adynamia, and hair loos, have been seen in only the DOX-treated group. Also, these animals were extremely anxious to all kinds of stimuli, especially 2 days after the application of DOX.p < 0.05) in comparison to control throughout the remaining 5 weeks of the study. The body weight of rats protected with AMI was also lower compared with the control group, especially during the last 5 weeks of experiment, but it was above that of the DOX-only treated group, although not significantly, especially during the last 3 weeks of the study.Rats treated with AMI-only and control group showed a steady weight gain, especially after the first week of treatment . AnimalsFour weeks after the last treatment, there were no statistical differences between the relative liver and kidney weights in rats pretreated with AMI, in comparison to control animals . HoweverMultiple applications of DOX decreased the total protein serum content in the rats 4 weeks after the last application, primarily as a result of a highly significant decrease of albumin . On the The significant increase of serum level of blood urea nitrogen and creatinine was observed in the group of rats treated with DOX . HoweverThe percentage of myeloblasts, promyelocytes, and myelocytes in the group of DOX-only treated rats was not significantly different from the control animals . HoweverThe relative number of lymphocytes has not been significantly changed in the bone marrow, although it has tendency to be decreased in all groups of treated rats in comparison to control, especially in the peripheral blood of rats pretreated with AMI .Also, pretreatment with AMI significantly decreased the percentage of eosinophil leucocytes in bone marrow, which was increased due to the effect of DOX . The relA careful analysis of the bone marrow in DOX-only treated rats showed marked eosinophilia and dyserythropoiesis, which was not present in AMI-pretreated animals A,B.Multiple applications of AMI-only affected neither of the examined hematological parameters, both in the bone marrow and peripheral blood, of the experimental animals 28 days after the last application of this protector.Microscopic examination of the hepatic tissue sections of the control animals has shown normal histological architecture without changes during the whole study period A. On theMicroscopic examination of the renal tissue sections of the control animals has shown normal histological architecture without changes A. On theSemiquantitative pathohistological analysis confirmed that the AMI given to rats as a pretreatment to DOX diminished degenerative and vascular changes in hepatic tissue caused by this antineoplastic agent, 4 weeks after the last treatment . In thisFour weeks after the last treatment, pretreatment with AMI significantly decreased the intensity of renal alterations caused by DOX . In thisOur results indicate that AMI, administered in a dose of 75 mg/kg, before each dose of DOX, significantly attenuated the effects of this antineoplastic agent  in rats. Moreover, the rats were monitored four weeks after the last administration of DOX, since our previous examination showed that the obtained survival of experimental animals  enabled further, more detailed research .We observed, as well as Rossi et al. , signifiWhen DOX was administered in small doses, over a period of weeks, depending on its cumulative dose, a significant decrease in the body weight compared to the control group was observed ,41. ThisIn addition, in the experiments in which AMI was used as a protector against toxic effects of DOX in mice, this protector reduced mortality and protected mice from the hematotoxicity of DOX . TherefoIn the bone marrow, good cellularity was established in all group of experimental animals, as well as the presence of individual hematopoiesis cell lines. Since the last administration of DOX was performed four weeks prior to the end of the study, there was sufficient time for bone marrow of animals treated with DOX to recover . HoweverThe pronounced monocytosis was established in the peripheral blood of both DOX only and AMI + DOX treated groups. This monocytosis could be interpreted as a possible recruitment of a monocyte–macrophage system for the elimination of DOX-affected tissues. For example, data on increased migration of macrophages into the thymus are known, in conditions such as increased destruction of thymocytes, due to the action of cyclophosphamide antineoplastic agent . Only thPrevious studies showed that pretreatment with AMI not only protected animals from the hematotoxicity of different antineoplastic agents ,45, but Serum biochemical examinations revealed that multiple application of DOX led to decrease in the serum total protein content, primarily albumin, and significant increases in blood urea nitrogen and creatinine content, while administration of AMI before DOX completely prevented these changes. The same biochemical profile of the results were obtained in DOX-induced nephropathy, as a model of chronic progressive glomerular disease ,58. Sign2, provokes hypoxic damage of the endothelial cells in capillary walls, which also contributes to further emerging of oedema [Moreover, pretreatment with AMI significantly reduced HDS and RDS in comparison to the rats given DOX only. It was shown that the earliest and most frequent changes in the rat liver and kidney after application of DOX, in cumulative dose, were the consequences of its toxic effect on the hepatocyte, endothelial cells, and renal tubular epithelial cells ,32,59. Tf oedema ,65. An af oedema ,32,59. SAlthough the exact mechanism of DOX-induced hepatotoxicity and nephrotoxicity remains unknown, it is believed that the toxic effects are mediated by free radical formation, iron-dependent oxidative damage of biological macromolecules, membrane lipid peroxidation, and protein oxidation ,26,66,672¯, including DOX-derived O2¯ generated by nicotinamide adenine dinucleotide (NADH) respiration of heart mitochondria particles [In AMI-protected animals, hepatic and renal tissue alterations were significantly less severe than those observed in animals treated with DOX only. It is well known that AMI active metabolite, WR-1065, once inside the cell, is powerful scavenger of oxygen free radicals ,34,35,36articles . Moreovearticles . This mearticles ,33, and articles  cardiotoarticles ,70,71. T®) for iv administration was purchased from Pharmacia & Upjohn . Amifostine was synthesized in the Chemical Department of Military Technical Institute, Belgrade, Serbia, by an original previously described procedure [DOX  with sawdust bedding  certificated as having contaminant levels below toxic concentrations. The environmental conditions were controlled and monitored by a central computer-assisted system with a temperature of 22 ± 2 °C, relative humidity of 55 ± 15%, 15–20 air changes/h, and artificial lighting of approximately 220 lux (12 h light/dark cycle). The experimental animals had free access to food, commercial pellets for rats  and tap water from municipal mains, filtered through 1.0 µm filter .Experiments were performed on male Wistar rats, 6–8 weeks old (200 to 220 g) bred at the Department for Experimental Animals, Military Medical Academy, Belgrade, Serbia. The experimental animals were housed in groups of five in plastic cages .Wistar rats were randomly divided into four experimental groups each containing 10 individuals. The animals received the following treatments: (1) Control (0.9% NaCl), (2) AMI (AMI 75 mg/kg ip) 30 min before saline (1 mL/kg ip), (3) DOX (DOX 1.25 mg/kg ip 4 times per week during 4 weeks), and (4) DOX (DOX 1.25 mg/kg ip 4 times per week during 4 weeks) + AMI (AMI 75 mg/kg ip 30 min before DOX). The total cumulative dose of DOX administered to rats (20 mg/kg ip) was estimated as being sufficient to induce progressive cardiomyopathy .Mortality and general condition of the animals were observed daily throughout the whole experiment lasting 8 weeks. Body weights were recorded 4 times per week for 4 weeks during the treatment, and once a week for a further 4 weeks, until the end of experiment. Postmortem examination, and liver and kidney excision and weighing, were also done at that time. Each experimental group consisted of 10 animals.The blood samples were taken and collected in plain tubes for serum separation. Then, blood samples were allowed to clot at room temperature (25 °C) for up to 30 min, after which they were centrifuged for 10 min at 3000 rpm at 25 °C. The separated serum was assessed for hemolysis, and then stored at −20 °C until assayed. The blood serum was analyzed by the auto-analyzer ASTRA-8  with the use of reagent kits. The standard parameters measured and methods adopted were as follows: total protein, albumin, blood urea nitrogen and creatinine, by end-point determination on colorimetric reaction.Twenty-eight days after the last DOX and/or AMI administration, ten animals from each experimental group were used for bone marrow and peripheral blood samples. For the preparation of the blood smear and differential blood count, blood was taken from tail vein of a rat by using syringes with needle. A small drop of the blood smears was prepared on clear glass slides, air-drying (2–4 h) at room temperature. Finally, slides were stained using routine May-Grünwald–Giemsa staining method.Then, animals were euthanized under light ether anesthesia. Both femurs of the rat were removed through the pelvic bone, and the femur bone was set free from the extra muscles. Then, epiphyses were cut off and bone marrow plugs were flushed out with a needle and aspirated into the syringe with small amount of fetal calf serum. The cell suspensions were centrifuged for 10 min at 1000 rpm, and sedimented cells were resuspended. A small drop of fine bone marrow cell smears was prepared from the final cell suspensions on clear glass slides. After air-drying (2–4 h) at room temperature, slides were stained using routine May-Grünwald–Giemsa staining method.Analysis of differential blood count and myelogram was done on the microscope  by using an immersion lens (1000×) and by counting at least 500 cells.The bone marrow was analyzed by determination of the quantitative relationship of granulopoiesis in comparison with erythropoiesis, as well as by the percentage representation of individual subpopulations of white blood cells. Also, a relative number of immature (myeloblasts and promyelocytes) and intermediate  leukocytes, between all experimental groups, was compared. The second group for comparison consisted of mature leukocytes (neutrophils and eosinophils). As a group called “eosinophils”, we considered cells with eosinophilic granules, immature as well as mature forms. Therefore, precursors of eosinophils have not been included in the subpopulation of myelocytes and metamyelocytes.In order to evaluate the hepatoprotective and nephroprotective effects of AMI, ten animals from each experimental group were sacrificed 28 days after receiving their last treatment under light ether anesthesia. At necropsy, the dissected liver and kidney tissue was carefully spread over a metal tray coated with wax and fixed with 10% neutral buffered formalin solution. Five to seven days after fixation, all tissues were divided into four portions in order to be prepared for making sections. After the fixation process, all tissue samples were dehydrated in graded alcohol , xylol, and embedded in paraffin blocks. Finally, 2 μm-thick paraffin sections were stained by hematoxylin and eosin (H&E) method, and whole visual fields magnified by 200× were analyzed .The type, degree, and severity of hepatic and renal lesions, along with the degree of inflammatory cellular infiltration, were assessed in all tissue sections from each animal, and they were counted in separate visual fields at 400× magnification . The severity of hepatic and renal lesions consisting of edema, cellular infiltration, hemorrhages, vacuolar degeneration, necrosis, and the distribution of lesions  were assessed and graded by two independent pathologists. From each slice, whole visual fields were analyzed by using light microscope according to the 5-point semiquantitative scale previously described ,73. A seU tests were used. All the analyses were estimated at minimal p < 0.05 level of statistical significance.Complete statistical analysis of data was done with the statistical software package . In the case of continuous data, variables were presented as mean value ± standard deviation (SD). Kolmogorov–Smirnov test was used for evaluation of normality of presented data. If data were normally distributed, 1-way ANOVA and post hoc Bonferroni tests were used. If data were not normally distributed, Kruskal–Wallis 1-way ANOVA and post hoc Mann–Whitney per se.Based on these findings, it can be concluded that AMI administered at a dose of 75 mg/kg 30 min before DOX significantly attenuated the effects of its cumulative dose (20 mg/kg body weight for 28 days) in rats, concerning animals’ mortality, body, liver and kidney weight, as well as structural alterations of the bone marrow, liver and kidney. In addition, this study showed that AMI, given in a dose of 75 mg/kg body weight, four times per week, for four weeks, is not toxic, Successful protection of rats by AMI against multiple-dose DOX-induced toxicity is mediated by complex, and still not fully elucidated mechanisms of action.


Giant HAAs (larger than 5 cm) are very rare. We present a case of an asymptomatic giant hepatic artery aneurysm (diameter 10.7 cm) discovered as an incidental finding on an  Visceral artery aneurysms comprise 1–2% of all vascular diseases . HepaticIn the majority of studies, the prevalence of HAAs accounts for both true and false aneurysms, but only a few studies distinguish between true aneurysms and pseudo-aneurysms. The majority of HAAs are true aneurysms that are extrahepatic and single ,10,11. TGiant HAAs (larger than 5 cm) are extremely rare, and very few cases have been reported ,15,16,1718F-fludeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) and confirmed with Doppler ultrasound.We present a case of a true, extrahepatic giant HAA, which was detected by 18F-FDG PET/CT for staging. 18F-FDG PET/CT was acquired 60 min after injection of 280 MBq 18F-FDG on a Biograph mCT Flow 64  with a flow table of 0.8 mm/s. The PET images were reconstructed using Ordered Subset Expectation Maximum, applying time-of-flight and point-spread-function. A CT (120 kV/10–150 mA) was performed without intravenous contrast due to the patient’s nephropathy.A previously healthy, 72 year old man with a history of well-regulated hypertension was diagnosed with bladder cancer causing severe nephropathy due to bilateral obstruction of the ureter ostium. The patient was referred to an 18F-FDG PET/CT revealed lymph node metastases and peritoneal carcinosis.Non-enhanced CT showed a large hollow process with a distinct calcified wall mimicking a porcelain gallbladder. However, the fused PET/CT demonstrated a discrete FDG uptake in the central part of the process, comparable to blood pool activity (SUVmax 3.5) with a surrounding mural thrombus  correspoThe patient was discussed at the urological multi-disciplinary team conference and, based on the disseminated urothelial carcinoma, no treatment with curative intent was possible. The patient was offered palliative chemotherapy. For evaluation and potential treatment of the giant HAA, the patient was referred to the Department of Vascular Surgery. Based on the poor prognosis of the cancer, it was decided that there was no indication for either open or endovascular treatment of the aneurysm due to the high risk of such procedures. Palliative treatment was initiated for the bladder cancer, and the patient was followed with non-contrast enhanced CT.A total of three CT scans were performed, showing minimal progression of the HAA from 107 × 105 mm to 110 × 106 mm. The patient did not experience any symptoms related to the aneurysm until his death 8 months later. The patient presented in this case report was identified during a quality assurance of our use of FDG PET/CT in bladder cancer. This study was approved by the Danish National Data Protection Agency and according to national legislation retrospective, observational studies do not require approval from the ethical committee.Owing to the increasing availability of imaging modalities over the last decades, the number of aneurysms detected prior to rupture has increased ,7. This Although the gold standard for diagnosing HAA is angiography ,15,18,19Doppler ultrasound is convenient for immediate confirmation of vascular lesions, determination of exact location and size of aneurysms, and differentiation from other vascular lesions, such as arteriovenous fistulas or vascular malformations . DopplerThe exact risk of rupture of a visceral aneurysm is unknown but has been reported to be between 14% and 80% . The prewww.vascular.org), no formal guidelines regarding visceral artery aneurysms exist, though some are under development. In the available literature, Abbas et al. [According to the Society of Vascular Surgery (s et al.  suggestes et al.  recommens et al. .In the present case, considering the patient’s short life expectancy, a conservative treatment course was adopted, and the patient’s aneurysm remained almost unchanged for 8 months.This present case highlights the ability of PET/CT to identify visceral aneurysms and to influence the diagnostic strategy and further treatment. Furthermore, due to the very rare frequency of giant HAAs and the absence of guidelines endorsed by internal societies of vascular surgery, the necessity of collecting multi-center data for the purpose of optimizing the management of patients with giant HAA should be addressed in the future.


Hsp) gene family members in the watermelon genome were identified and characterized by bioinformatics analysis. In addition, expression profiles of genes under combined drought and heat stress conditions were experimentally analyzed. In the watermelon genome, 39 genes belonging to the sHsp family, 101 genes belonging to the Hsp40 family, 23 genes belonging to the Hsp60 family, 12 genes belonging to the Hsp70 family, 6 genes belonging to the Hsp90 family, and 102 genes belonging to the Hsp100 family were found. It was also observed that the proteins in the same cluster in the phylogenetic trees had similar motif patterns. When the estimated 3-dimensional structures of the Hsp proteins were examined, it was determined that the α-helical structure was dominant in almost all families. The most orthologous relationship appeared to be between watermelon, soybean, and poplar in the ClaHsp gene families. For tissue-specific gene expression analysis under combined stress conditions, expression analysis of one representative Hsp gene each from root, stem, leaf, and shoot tissues was performed by real-time PCR. A significant increase was detected usually at 30 min in almost all tissues. This study provides extensive information for watermelon Hsps, and can enhance our knowledge about the relationships between Hsp genes and combined stresses. Heat shock protein ( Citrillus lanatus (watermelon) is an important plant in the family Cucurbitaceae, constituting 7% of the world’s total fields devoted to crop production. The world annual watermelon (Citrillus lanatus) production is about 90 million tons, and watermelon (Citrillus lanatus) is among the 5 most consumed fresh fruits . Although watermelon (Citrillus lanatus) is mostly composed of water (up to 90%), it contains important nutritious compounds such as sugar, lycopene, and health-promoting amino acids including citrulline, arginine, and glutathione . Heat-shock proteins (Hsps) are a special protein family that are synthesized in response to a variety of stress conditions including high temperatures, and are required for the growth and survival of the cell . Hsps are functional in many processes such as protein folding, cellular regulation, and inhibition of the accumulation of inappropriate proteins in the cell. In addition, members of this protein family are known to be synthesized under different stress conditions and behave like molecular chaperones, which enable proteins to become three-dimensional structure by folding . They are also key determinants of quality control and have an important role in protecting overall cellular protein balance .Hsps with molecular weights ranging from 10 kDa to 200 kDa are classified according to their molecular weights and functioning mechanisms. They can be divided into 6 classes: sHsp , Hsp40 (J-proteins), Hsp60 (chaperonins), Hsp70, Hsp90, and Hsp100 (Clp proteins). sHSPs that are not present in plant cells under physiological conditions are stimulated by stresses including drought, salinity, oxida species, and low temperatures . Hsp40 is characterized by a standard J-domain. J-protein heat shock family members are found everywhere in prokaryotes and eukaryotes at various subcellular sites. J-proteins stand for a large family of molecular chaperones that serve to determine the cellular functions of Hsp70s . The Hsp60 family, found in plastids, mitochondria, and cytoplasm of all eukaryotes and eubacteria, are molecular chaperones that are among the most conserved and found everywhere . Hsp70 is involved in various cellular functions such as protein transport between membranes, modulation of protein activity, protein folding, regulation of protein degradation, and inhibition of irreversible protein aggregation . Hsp90 is one of the most abundant proteins in eukaryotes. Unlike other chaperones, Hsp90 is not effective for new protein folding. At a later stage of folding, it binds to substrate proteins that are close to natural . Hsp100/Clp is a class of molecular chaperones that have the ability to resolve almost every protein which has collected after severe stress. They are not necessary under normal growth conditions, and are triggered by extreme heat or other severe stresses .Under both natural and agricultural conditions, plants are often exposed to environmental stresses . Abiotic stresses including drought, salinity, extreme temperatures, chemical toxicity, and oxidative stress create serious threats to agriculture and cause deterioration of the natural environment . Drought is one of the most significant abiotic stress factors that decreases crop yield with soil yield . Drought causes the stoma closure associated with reduced CO2 intake in leaves; this results in instabilities between photosynthetic electron transports and carbon assimilation . High temperature influences the metabolism and structure of plants, especially cell membranes of plants, and many basic physiological processes including photosynthesis, respiration, and water relations . The combination of drought and heat stress has been found to have more detrimental effects on the growth and productivity of plants compared to each of the different stresses applied separately. For example, when plants encounter heat stress, they open their stomata to cool their leaves by transpiration. When heat stress is combined with drought, the temperature of the leaves is higher because the plants cannot open their stomata. Similar problems will occur when salinity or heavy metal stress is combined with heat stress, because more transpiration leads to more salt or heavy metal intake. Since most of the abiotic stress studies performed in laboratory conditions do not fully reflect the conditions of the natural environment, there may be differences between the results of these studies and the conditions required for the development of highly tolerant plants in natural conditions. This difference explains why transgenic plants with high tolerance to abiotic and biotic stress under laboratory conditions may not show high tolerance in the natural environment. Molecular, physiological, and metabolic examination of the combination of stressors facilitates the elimination of this difference and the development of plants that are more tolerant to natural stress conditions . Since plants in their natural environment are often exposed to combined stresses such as drought and high temperature, researching the response of plants to combinations of stress factors can help resolve stress tolerance mechanisms in plants.Arabidopsis , wheat , sunflower , rice , tomato , poplar , and eucalyptus . The complete genome sequence of watermelon was published by Guo et al. in 2013; however, to our knowledge, Hsp family members have not yet been defined in the watermelon genome. In the current study, watermelon Hsp genes have been identified and characterized. In addition, expression profiles of genes under combined drought and heat stress conditions were experimentally analyzed and the results were compared using bioinformatics.Complete genomic data of organisms are useful for determining important gene families using bioinformatics methods. Hsps have been characterized in many plant species, including Hsp genes from the watermelon genome. First, using the HSPIR (Heat Shock Protein Information Resource) database, the genomic, coding region, and protein sequences of Hsp genes were obtained for all plants. For these sequences, a BLASTP (Protein Blast Sequence Comparison) search was performed using the Cucurbit Genomics database. In addition, defined sequences were scanned and selected according to the protected regions by using the Hidden Markov Model (HMM). Protected regions of the selected sequences were checked with the Pfam (https://pfam.xfam.org/) database, and they were retrieved for the study as watermelon Hsps.Instability index, molecular weights, and isoelectronic effect values (pI) of these Hsp sequences were obtained using the ProtParam tool (u1d45).According to our previously published study , different search strategies were performed to elucidate Hsp genes found in watermelon plants were made using the Cucurbit Genomics database (u1d46); these localizations were then shown on chromosomes with the MapChart program. To clarify the structure of the watermelon Hsp genes, the exon–intron regions of the genes were determined by using the Gene Structure Display Server (http://gsds.cbi.pku.edu.cn/) .Chromosomal localizations of the Multiple sequence alignment of the defined watermelon Hsps was performed using ClustalW via the MEGA7 program. Phylogenetic trees were drawn using sequences aligned with the ClustalW program. Maximum likelihood method  was used when the phylogenetic tree was drawn. Through this method, a phylogenetic tree was formed with 1000 repetitive bootstrap analyses. The drawn phylogenetic tree was visualized using the iTOL database, and each cluster was indicated in a different color. The MEME Suit web portal  was used to identify the preserved motifs. When defined, the number of motifs was 20 and the width of the motif was chosen as optimum ≥2 and ≤300. The obtained motifs were also scanned using the InterPro database with InterProScan .The Blast2Go program was used for functional analysis of determined watermelon Hsps . The amino acid sequences of the watermelon Hsps were loaded, and biological and molecular functions and cellular contents were obtained by GO classification by following steps such as BLAST, interpro, mapping, annotation, charting, and graphing.Hsp genes in the watermelon genome and Arabidopsis thaliana, Oryza sativa, Glycine max, Populus trichocarpa, Vitis vinifera,and Zea mays. Afterwards, homologous (Ks) and nonhomologous (Ka) exchange rates were calculated using the PAL2NAL program by a method which aligns the amino acid sequences of the Hsp genes with their original complementary DNA sequences . Thus, for each Hsp gene, duplications and separation time  were calculated using homologous mutation ratios of λ changes corresponding to each homologous region and year (T = Ks/2λ (λ = 6.5 × 10−9) .Multiple amino-acid sequence alignment was performed through the ClustalW program by aligning orthologous gene pairs among the duplicated A Protein Data Bank (PDB) scan was performed to identify the sequences which were similar to watermelon Hsp proteins and the most suitable sample with a known 3-dimensional structure by using BLASTP . Possible 3-dimensional structures of the watermelon Hsps were analyzed by using the Phyre2 program with the obtained data .Hsp gene transcripts in the watermelon plant using the psRNA Target server (http://plantgrn.noble.org/psRNATarget/home) .The plant miRNA database was used to identify miRNAs targeting previously known plant miRNA genes using the miRBase v20.0 (http://www.mirbase.org/) program for the identification of miRNA-controlled gene targets . All assumed plant and watermelon miRNAs were identified by aligning all known plant miRNAs with For RNA-Seq analysis, all Illimuna HiSeq readings were provided through an open database archive called SRA (Sequence Read Archive). Accession numbers: SRR1724899, SRR1724900, SRR1724901, SRR1724902, SRR1724903, SRR1724943 , WM-UR-1/SRR1001435, WM-UR-2/SRR1001436 (white fruit 10 days after pollination), WM-IM-1/SRR1001437, WM-IM-2/SRR1001438 (white–pink fruit 18 days after pollination), WM-PM-1/SRR1001439, WM-PM-2/SRR1001440 (pink fruit 28 days after pollination), WM-MA-1/SRR1001441, WM-MA-2/SRR1001442 (ripe red fruit 34 days after pollination), SRR494474, SRR518988, SRR518988 (phloem tissue), SRR494479, SRR518992, SRR518993 (vascular tissues). The “.sra” raw sequence data of all readings was downloaded and converted to the “fastq” format using the NCBI SRA Toolkit’s fastq-dump command. FastQC analysis was carried out in order to perform quality control on all of the remaining readings to remove the low-quality ones from the obtained readings. Normalization and transformation of all readings were performed with the CLC Genomic Workbench version 11.1 program. A hierarchical clustering map (HeatMap) was drawn using gene expression measurements with the Permut Matrix program.Watermelon seeds (Crisby F1) were used in the experiments. These seeds, which were supplied by Monsanto Food and Agriculture Trade Limited Company , were washed and kept in distilled water for 2 h. At the end of the 2 h, the seeds were planted in plastic pots containing vermiculite. Diameter and volume of plastic pots used in the study were 93 mm and 400 cc, respectively. After the planting was completed, the pots were irrigated with Hoagland solution  and transferred to the climate cabinet. They were grown at 24 ± 2 °C with a 16 h light and 8 h dark photoperiod in the climate cabinet for approximately 35 days at a light intensity of 400 μmol m–2 s–1, and with relative humidity maintained at 55%–80% by watering with Hoagland solution regularly every day. Drought and temperature stresses were applied to the plants together to see their combined effect. A 20% Hoagland solution containing polyethylene glycol 6000 (PEG-6000) was used for drought stress. For temperature stress, the temperature of the climate cabinet was set at 50 °C. The samples were collected at 0, 30 min, 1 h, and 2 h of stress application as in the study conducted by Baloğlu et al. . Samples collected at the zero hour were used as controls. Four different tissue types were collected from the plants. They were root, stem, leaf, and tendril. All leaves except the first-grown cotyledon leaves were collected for analysis. All of the roots and tendrils that could be obtained were collected. All collected samples were frozen in liquid nitrogen and stored at –80 oC until RNA isolation.Hsp genes were determined based on their expression patterns in transcriptome data and were designed using the NCBI Primer Blast tool (u1d48) . Real-time polymerase chain reaction (qRT-PCR) was achieved with a Qiagen Rotor Gene 6000 “Real-Time” PCR device. For reaction, temperature optimized primers and SYBR Green Supermix  were used. From the cDNA samples, 6 trials  were used. The components for qRT-PCR reaction were 20 μL in total, including 1 μL forward primer, 1 μL reverse primer (25 pmol/μL), 2 μL cDNA (100 ng/μL), 10 μL SYBR Green Supermix, and water. qRT-PCR conditions were as follows: after first denaturation at 95 °C for 5 min, 1 min at 95 °C, 1 min at 55–58 °C, and 1 min at 72 °C were repeated 35 times. The amplified cDNAs were analyzed by qRT-PCR at 530 nm. In addition, melting curve analysis was performed to verify the correct replication. In qRT-PCR analysis, a reference gene that is known to be unchanged or least affected in expression under various conditions was used for normalization. Tua gene  was chosen as a reference gene in the study . Calculated ΔCT (ΔCT = CT sample – CT reference) and ΔΔCT (ΔΔCT = ΔCT stressed sample – ΔCT control) values were used to reveal the differences in gene expression levels, as 2ΔΔCT and graphs of expression profiles were drawn .Total RNA was extracted using a Trizol reagent  procedure which has been previously described in our studies . Absorbance values were then measured with a spectrophotometer  at 260/280 nm wavelengths in order to control the purity and quality of RNA dissolved in DEPC water. DNase  was applied for the purification of the isolated RNA samples completely from DNA residues. An u1d47, Hercules, CA, USA) was used for cDNA synthesis. Primers for = <0.05; ***, P = <0.01. Using the univariate-ANOVA test for statistical analysis of the results, it was accepted that the difference was significant when P value was <0.01.Statistical calculations and comparisons were made by using the Minitab 18 package program. The difference between stressed samples and control samples is shown as follows: *, P = <0.1; **, PClasHsp-01 to ClasHsp-39. One hundred and one genes for Hsp40 (ClaHsp40), 23 genes for Hsp60 (ClaHsp60), 12 genes for Hsp70 (ClaHsp70), 6 genes for Hsp90 (ClaHsp90), and 102 genes for Hsp100 (ClaHsp100) were determined and numbered according to their chromosomal localization. It was determined that the length of the sHsps changed between 97 and 509 aa; most of them were acidic. The length of Hsp40 proteins changed between 60 and 2548 aa; 62 of the proteins were basic and 39 were acidic. It was seen that the protein lengths of the Hsp60s ranged from 57 to 1741 aa; 17 were acidic, 6 were basic. Amino acid content of Hsp70 proteins varied from 571 to 898 aa; all of them were acidic. It was found that the protein length of the Hsp90s was 699 to 811 aa long; all of them were acidic. Hsp100 proteins were 149 to 1911 aa in length, and 66 were acidic. The acidic property was dominant among Hsps when the results were considered. This can be attributed to the roles of Hsps in the cell. Detailed information including chromosomal localization, molecular weight, and instability index of watermelon Hsps are given in Supplementary material 2.Hsp sequences from various plant species were obtained from the HSPIR database, and those belonging to watermelon plants were identified using the Cucurbit Genomics database. As a result, 39 genes belonging to sHsp were found and named, from ClasHsp genes (10) were located on chromosome 7. There were 5 genes on the fifth and tenth chromosomes. When the chromosomal distribution of the ClaHsp40 genes was examined, it was determined that the most genes (14 genes) were located on chromosomes 2 and 10, followed by chromosomes 5 and 9 with 12 genes . When the location of the ClaHsp60 genes on the watermelon chromosomes was examined, 5 of the Hsp60 genes were located on chromosome 11. The third, sixth, and ninth chromosomes had 3 ClaHsp60 genes, the fifth and tenth chromosomes had 2 ClaHsp60 genes, and other chromosomes had 1 each of the ClaHsp60 genes . The ClaHsp70 genes were located on chromosomes 1, 4, 5, 6, 9, 10, and 11. It appeared that the ClaHsp90 genes were located on chromosomes 1, 2, 3, and 8. The majority of the ClaHsp100 genes (19 of the ClaHsp100 genes) were located on chromosome 1.The genes identified for the Hsp family were distributed over 11 chromosomes in the watermelon . The majority of ClaHsp genes were examined, it appeared that the exon and intron regions were approximately equal in sHsp, Hsp40, and Hsp70 genes. Hsp60 and Hsp90 genes were found to have entirely exon regions and no intronic regions. Hsp100 genes had very few introns, mostly exons. Similarly, Arabidopsis, castor bean, rice, and sorghum were observed to have the same characteristics , suggesting evolutionary conservation. This can be attributed to their many functions in the cell. The exon–intron structures of the ClaHsps are shown in Supplementary material 3.When the exon–intron structures of the analyzed A phylogenetic tree was drawn and motif analysis was performed to evaluate the evolutionary relationships between the ClaHsps. When the phylogenetic trees drawn by maximum likelihood method were examined, it was seen that sHsp proteins divided into 4 main clusters (Clusters I–IV). It was determined that each cluster contained 7, 15, 6, and 11 proteins, respectively. The phylogenetic tree of ClaHsp40 proteins included 5 main clusters; 3, 3, 2, 5, and 88 proteins were detected in each cluster, respectively. The phylogenetic tree of ClaHsp60 proteins had 6 main clusters. According to phylogenetic analysis of ClaHsp70 proteins, 6 main clusters were formed. Four main clusters were defined for ClaHsp90 proteins. The ClaHsp100 proteins were divided into 6 main clusters in the phylogenetic tree. As a result, 4, 5, 7, 12, 36, and 38 proteins were found in each cluster, respectively . Each Hsp family was divided into a different number of subgroups . The protected motifs obtained from the MEME suite database were analyzed. A total of 20 different motifs were identified for each Hsp family. When the motifs of ClasHsps were examined, 3 dominant motif patterns were observed. Seven motif patterns were determined among ClaHsp40 proteins. When the motif structure of ClaHsp60 was analyzed, the same motif structures were included in the proteins. Four motif patterns were shared among ClaHsp70 proteins, whereas ClaHsp90 proteins had 14 motif patterns. ClaHsp100 proteins had 6 dominant motif patterns. As a result, it has been determined that these genes contain motif patterns in accordance with the constructed phylogenetic trees .Hsp genes were determined by gene ontology analysis . sHsps have roles in cellular functions and response to stimuli, and are mainly found in the membrane, cell, and cell parts. It has been determined that Hsp40 proteins have variable biological functions such as regulation and positive regulation of biological processes, multiorganism processes, reproduction, multicellular organismal processes, biological regulation, cellular component organization, and biogenesis; the molecular function is binding and catalytic activity. ClaHsp40 family members were especially found in the organelles, membranes, cell, and cell parts. The Hsp60 ontology analysis showed that their biological function was mostly in cellular processes. Hsp60 was found to be the most abundant in the cell and cell parts. Hsp70 proteins had roles in cellular functions, metabolic processes, and single organism processes. They were located especially in the cell, cell parts, and organelles. Hsp90 proteins had 2 functions as a response to stimuli and cellular processes. Many biological processes including regulation of biological processes, response to stimuli, cellular component organization and biogenesis, cellular functions, and metabolic functions were determined for Hsp100 proteins. It was found that this protein group was mostly located in the membrane, cell parts, and organelles within the cell. It was observed that the molecular function of all ClaHsp proteins was achieved by binding activity.Biological function, cellular localization, and molecular function of watermelon Hsp gene families which were obtained from the watermelon genome were compared with sequences of different organisms , and orthologous analysis was performed. As a result, orthologous gene pairs, similar and dissimilar rates of change, and probable differentiation times in the evolutionary process have been determined . According to the obtained data, the most orthologous relationship for the ClasHsp gene family was found to be with soybean (47 pairs) followed by poplar (35 pairs). The least orthologous relationships were observed with grape (5 pairs) and maize (6 pairs). The organisms with the highest similarity to watermelon among the 6 organisms for this gene family were soybean (0.05), grape (0.05), and poplar (0.05). The organism with the lowest similarity was identified as maize (0.02). When the divergence times were considered, it was determined that the first divergence with watermelon was by Arabidopsis, with an average of 227 million years ago (MYA). It was determined that the organism that last diverged from watermelon was grape, with 18 MYA for sHsp genes . Sequences of ClaHsp40 gene family and Hsp40 genes from other plants were examined, it was observed that the most orthologous relationships were with soybean (419 pairs) and poplar (338 pairs). The least orthologous relationships with watermelon Hsp40 genes were found with rice (173 pairs) and grape (124 pairs). The highest similarity ratio was found with soybean (0.11) and poplar (0.10) Hsp40 genes. The least similarity was determined between rice and watermelon with 0.03 similarities. When the possible separation times were examined, it was observed that the first separation was with rice about 204 MYA. It was then separated from maize about 198 MYA. It was observed that the latest separation was with poplar, about 72 MYA .When the orthologous relationships between the ClaHsp60 gene family were examined, it was observed that the most orthologous relationship was with soybean, with 72 pairs. This was followed by poplar with 64 pairs. The orthologous relationships with maize and Arabidopsis included 41 and 40 pairs, respectively. In the ClaHsp60 genes, the organisms which had the least orthologous relationship with watermelon were rice and grape with 37 and 30 pairs, respectively. Grape had the highest similarity ratio with watermelon with 0.05. The similarity ratio between watermelon and soybean was 0.04. This was followed by Arabidopsis and poplar with 0.03 and 0.25, respectively. The organisms which had the lowest similarity ratio with watermelon were rice and maize with 0.02. When the estimated divergence times with these organisms were considered, the first separation was with maize 157 MYA. It was observed that the last separation was with soybean and poplar about 51 MYA .When the orthologous relationships of the ClaHsp70 gene family were evaluated, it was determined that the most orthologous relationship was between watermelon and soybean (148 pairs). This relationship was followed by poplar with 113 pairs. The least orthologous relationship was between watermelon and grape with 28 pairs. When the similarity rates with these organisms were examined, Arabidopsis, rice, and maize were found to have almost the same similarity ratio (0.01). The grape had the highest similarity ratio (0.03). Considering divergence times, the earliest diverged genes from watermelon were rice genes, with an average of 275 MYA. The latest divergence was determined to be with poplar, 112 MYA .When the orthologous relationships of the ClaHsp90 genes were analyzed, the most orthologous relationship was with poplar with 78 pairs. This was followed by soybean, with a highly orthologous relationship with 52 pairs. The least orthologous relationship was found with grape (11 pairs). When similarity ratios were examined, it was determined that watermelon and Arabidopsis, soybean, grape, and poplar had almost the same similarity rate. Rice and maize had about the same similarity ratio with watermelon. Considering divergence times of ClaHsp90 genes, the first diverged genes from ClaHsp90 genes were maize genes, with an average of 153 MYA. It was estimated that the most recent divergence was with poplar 49 MYA .When the orthologous relationships of ClaHsp100 genes with other organisms, it was found that the most orthologous relationships werebetween watermelon and soybean (259 pairs) and watermelon and poplar (189 pairs). These were followed by maize (126 pairs) and Arabidopsis (123 pairs). The least orthologous relationships were with grape and rice with 100 and 98 gene pairs, respectively. When the similarity ratios of these organisms with watermelon were calculated, it was determined that grape and poplar had the highest similarity ratio (0.06). It was then observed that similarity rates for soybean and Arabidopsis were 0.05 and 0.04, respectively. The lowest similarity ratio was found to be with rice and maize (0.03). When the estimated divergence times were examined, the first separation was with maize (83 MYA). The latest organism to separate from watermelon was grape (25 MYA)  .Finally, when considering the orthologous relationships of Information about the predicated 3-dimensional structures of ClaHsps were obtained by homology modeling using the Protein Data Bank (PDB). When sHsps were examined, 20 ClasHsp proteins showed high similarity. The similarity rate was determined by selecting the intensive mode from the Phyre2 database. Reliability was 90%, and similarity was between 70% and 100%. As a result, it was seen that β-sheet structure predominated in the 3D structures of ClasHsps. When the other Hsp family members were analyzed, 4 proteins for the ClaHsp40 family, 15 proteins for the ClaHsp60 family, 9 proteins for the Hsp70 family, 3 proteins for the Hsp90 family, and 38 proteins for the Hsp100 family showed high similarity. Alpha helix structure was dominant among all of these analyzed proteins, and a few β-sheet structures were determined, except for ClaHsp40 proteins. Detailed figures are given in the Supplementary material 7.Hsp genes were targeted by miRNAs. ClasHsp-11, ClaHsp40-05, ClaHsp40-06, ClaHsp40-30, ClaHsp40-31, ClaHsp60-01, ClaHsp60-10, ClaHsp60-12, ClaHsp70-07, ClaHsp100-05, ClaHsp100-11, ClaHsp100-15, ClaHsp100-16, ClaHsp100-58, ClaHsp100-85, ClaHsp100-86, ClaHsp100-91, ClaHsp100-96, and ClaHsp100-97 genes were targeted by a total of 23 different miRNAs. ClaHsp genes targeted by miRNAs are shown in detail in Figure 5.As a result of miRNA analysis using the psRNA Target Server (http://plantgrn.noble.org/psRNATarget/), it was determined that a total of 19 different watermelon Hsp genes, individual heat maps for each Hsp family were drawn using SRR files. As a result of SRA analysis, expression levels under various stress conditions in different tissues of the watermelon were examined. Heat maps containing different tissues and fruits are shown in Supplementary material 8. According to the results, ClasHsp-23 gene was upregulated in thefloem and vascular tissues and on the 10th, 18th, 26th, 28th, and 34th days of fruit development after pollination. There was an increasing pattern in the expressions of ClaHsp40-15 and ClaHsp40-96 genes on the 18th, 26th, 28th, 34th, 42nd, and 50th days of fruit development stages, as well as in vascular and floem tissues. Additionally, ClaHsp60-09 and ClaHsp60-15 genes have an increase profile in all studied stages of fruit development. In addition, ClaHsp70-07, ClaHsp70-10, ClaHsp90-04,and ClaHsp90-05 genes were upregulated on the 18th, 26th, 28th, 34th, 42nd, and 50th days of fruit development and in vascular and floem tissues. Moreover, the expressions of ClaHsp100-54 and ClaHsp100-67 genes were induced on the 18th, 28th, 34th, 42nd, and 50th days of fruit development stages and in vascular and floem tissues. To determine the expression of the watermelon Hsp genes from watermelon using qRT-PCR. For each Hsp family, qRT-PCR analyses were performed using root, stem, leaf, and tendril samples, and time-dependent expression graphs were drawn . When the expression levels of the watermelon stem sample were examined, it was observed that there was an increase pattern at 30 min of combined stress application in the expression levels of all of the Hsp families . Subsequently, it was determined that the expressions levels decreased during the first hour and increased again during the second hour. Based on the transcriptome results, combined drought and temperature stress responses were tested for in selected ClasHsp-23 gene from the first hour. A significant increase was observed for the ClaHsp40-15, ClaHsp60-15, and ClaHsp100-67 genes for the first 30 min, and a decrease in the following hours was determined. It was also observed that the maximum expression for the ClaHsp60-15 and ClaHsp100-67 genes was at 30 min. For the ClaHsp70-07 and ClaHsp90-04 genes, it was determined that the first reaction to stress was in the first hour. When watermelon tendril samples were viewed, it was detected that the expressions of the ClasHsp-23, ClaHsp60-15, ClaHsp70-07, ClaHsp90-04, and ClaHsp100-67 genes were upregulated at 30 min, then downregulated at 1 h, and increased again at 2 h. In the tendril samples, the highest response against combined drought and temperature stress was observed at 30 min for the ClaHsp70-07 and ClaHsp90-04 genes.When the watermelon leaf sample was examined, it was observed that there was a certain increase in the ClaHsp40-15, ClaHsp90-04, and ClaHsp100-67 genes up to 30 min and then the expression was decreased. It was determined that in the ClasHsp-23 and ClaHsp70-07 genes, expression increased at 30 min, followed by a decrease in the first hour and a rise in the second hour. The highest expression level in the ClaHsp60-15 gene was observed at the first hour. When the watermelon root samples were examined, it was observed that there was an increase pattern in the ClasHsp-23, ClaHsp60-15, ClaHsp70-07, ClaHsp90-04,and ClaHsp100-67 genes. In stressed root samples, this period was observed in the ClasHsp-23 and ClaHsp70-07 genes. The ClasHsp-23 and ClaHsp70-07 genes can be described as the first genes to respond to the stress in the stem, root, and tendril tissues. In addition, ClaHsp100-67 gene expression showed a rapid increase in the first 30 min of the stress application in all studied tissues. For this reason, the ClaHsp100-67 gene may be considered an early response gene which responds to combined drought and temperature stresses in all tissues .When all the results were evaluated, a rapid increase pattern was seen in the first 30 min of combined drought and temperature stresses. The expression levels of all of the genes studied in the watermelon stem tissues thereafter decreased at the first hour and increased again in the second hour. In the tendril tissue samples, this expression period was commonly observed in Researching the response of plants to a combination of different stresses can be helpful for elucidating stress response mechanisms in plants, because plants are often exposed to a combination of stresses in nature, including drought and heat shock. Hsp genes against high temperature and drought stress combinations were examined, and the response to these stresses was interpreted. As a result of bioinformatics analysis, genes belonging to each Hsp family were determined in the watermelon genome. The Hsp40 and Hsp100 families had the highest number of genes with 101 and 102 genes, respectively. After that, 39 genes belonging to the sHsp family and 23 genes belonging to the Hsp60 family were found. The Hsp families containing the lowest number of genes in the watermelon genome were Hsp70 and Hsp90 with 12 and 6 genes, respectively.In this study, the changes in the expression profiles of watermelon ClaHsp genes were identified in the watermelon genome, located over 11 watermelon chromosomes. The most ClaHsp genes were on the fifth chromosome, and the lowest number of Hsp genes were on the eleventh chromosome. The genes belonging to the Hsp family in the watermelon genome have been identified for the first time in this study. In the literature, similar gene numbers have been observed in other organisms when compared to the Hsp genes detected in the watermelon genome. In a study conducted by our research group, it was determined that 60 sHsp, 145 Hsp40, 49 Hsp60, 34 Hsp70, 12 Hsp90, and 90 Hsp100 genes were identified in the poplar genome . Members of the Hsp70 family are the most thoroughly studied group of stress proteins up to the present. As a result, 32 genes in the rice , 21 genes in the eucalyptus , 18 genes in the Arabidopsis , 13 genes in the beech, 17 genes in the oak, and 15 genes in the chestnut  genomes have been identified as Hsp70 genes. Other studies on other Hsp family members have identified 29 Hsp60 genes , 7 Hsp90 genes , and 13 sHsp genes  when researching the Arabidopsis thaliana genome. Furthermore, 23 sHsps , 104 Hsp40 , 20 Hsp60 , 9 Hsp90, and 10 Hsp100  proteins were determined in the rice genome. According to our results, the most numerous Hsp family members in the watermelon genome were Hsp40 and Hsp100. This was similar with poplar for Hsp40 and Hsp100, and rice for Hsp40. Moreover, Hsp100/ClpB proteins have an important role in tolerance to high temperatures in various organisms . Therefore, it is an expected situation to have a large number of Hsp100 genes in watermelon growing in a hot climate. It is an expected situation that the number of Hsp40 proteins acting as cochaperones of the Hsp70 protein is higher than that of Hsp70. A total of 283 ClaHsp genes. In the ClasHsp, ClaHsp40, and ClaHsp70 families, exons–introns were approximately equal, while very few introns were detected in ClaHsp100 and no introns were observed in the ClaHsp60 and ClaHsp90 families. When the other studies were examined, it was seen that the exon structure predominated in the sHsp, Hsp70, and Hsp100 genes of the Hsp families in the A. thaliana genome . When the Hsp70 family in the eucalyptus genome was examined, it was found that there were an approximately equal number of exon–intron structures as in the watermelon Hsp70 family . Genes with the same physical structure, such as exon–intron length, were located in the same cluster in the phylogenetic tree. It can be concluded from this that the phylogenetic tree distribution in each cluster is consistent with the exon–intron structures. Exon–intron structures have been determined in order to examine the structure of the Molecular and biological functions and localization of every Hsp family in the cell were determined by analyzing the gene ontology of ClaHsps. As a result, it was seen that the molecular function of each Hsp family was predominantly binding. Their biological functions were often as a response to stimuli; they were also involved in cellular and metabolic processes and the regulation of biological functions. It was observed that ClaHsps were localized in different parts of the cell. They were mainly found in the inner parts of the cell, membranes, and organelles. When other studies in the literature were examined, it was found that Hsp families were mostly located in intracellular parts in eucalyptus  and poplar . The results for watermelon Hsp proteins have parallels with the results of these studies. These localizations may be the reason for the multiple roles of these family members in organisms. Hsp genes in the watermelon genome with those of other organisms, we have identified orthologous genes that are different in structure but functionally similar. We gathered information about the evolutionary processes of ClaHsp genes and the orthologous relationships between watermelon and Arabidopsis, soybean, rice, poplar, grape, and maize. In the majority of ClaHsp gene families apart from ClaHsp90, the most orthologous relationship appeared to be between watermelon and soybean; this was closely followed by poplar. For ClaHsp90 genes, the most orthologous genes were defined with poplar and then with soybean. The least orthologous relationship was found between watermelon and grape. When the similarity rates were examined, high similarity was observed between watermelon and soybean and grape Hsp genes. It has been determined that maize had the least similarity with watermelon. The first organism that separated from watermelon was usually maize or rice for the ClaHsp gene families. When the results of these analyses were considered, it was thought that Hsp genes in the watermelon and in the poplar diverged later than the Hsp genes in other organisms. Therefore, the differentiation was lower with the poplar. Another study which reported the high similarity between the watermelon and the poplar LEA genes was also performed by our group. It has also been observed that the most recent divergence time for LEA genes was between watermelon and poplar . In addition, the earliest divergence of Hsp genes in the poplar genome was determined with the rice genome, as for many watermelon Hsp genes . These results may help achieve better understanding of the evolutionary relationship between these organisms and the watermelon based on the Hsp genes. Therefore, it is thought that more efficient results can be obtained in future plant breeding and cloning studies by using this evolutionary relationship. By comparing the This structural feature could allow the proteins to bind firmly to the substrate binding domain (SBD). It could be said that α-helix and β structures are dominant in molecular functions and that therefore they are an important form of structure. The determination of these structural properties provides an idea for determining the structure, function, and mutation relationships of proteins . In addition, this structure may contribute to fluid loss and alleviate potential damage in plant wounding or stress .When the predicated 3D structure of proteins was examined, it was seen that β-sheet structure predominated in ClasHsps. However, the alpha helix structure was dominant among all other analyzed proteins and a few β-sheet structure were determined, except in ClaHsp40 proteins.Hsp genes in the watermelon genome were examined, the miRNAs targeting the ClasHsp-11 gene were mtr-miR2673a and mtr-miR2673b. In the literature, miR2673 appeared as a miRNA associated with drought . In the ClaHsp40 family, ClaHsp40-05, ClaHsp40-06, ClaHsp40-30, and ClaHsp40-31 genes were targeted by ath-miR870-5p, bdi-miR7787-5p, stu-miR8046-3p, sbi-miR529, and osa-miR529a miRNAs. The ClaHsp60-01, ClaHsp60-10, and ClaHsp60-12 genes of the ClaHsp60 family were targeted by osa-miR5522, ata-miR172a-5p, and ath-miR414 miRNAs. It has been determined that miR172 is associated with drought stress and the coding of transcription factors for flower development in Arabidopsis. Therefore, it has been concluded that miRNA plays significant roles in plant growth, metabolism, and response to stress . In addition, other studies for miR414 have found that miR414 has an important role in the regulation of plant growth and development . It was determined that the miRNA targeting the ClaHsp70-07 gene from the ClaHsp70 family was OsA-miR2875. We have not found an miRNA that targets genes belonging to the ClaHsp90 family. ClaHsp100-05, ClaHsp100-11, ClaHsp100-15, ClaHsp100-16, ClaHsp100-58, ClaHsp100-85, ClaHsp100-86, ClaHsp100-91, ClaHsp100-96, and ClaHsp100-97 genes were targeted by miR6473, miR1311, miR414, miR2673, miR8576, miR5209, miR9778, miR167i, miR5658, and miR614c miRNAs. The ClaHsp100 family had the Hsp genes most often targeted by miRNAs. In total, 19 different watermelon Hsp genes were targeted by 23 different miRNAs. Determination of miRNAs targeting ClaHsp genes can help to resolve their functions in the watermelon.When miRNAs targeting these Hsp genes were examined by qRT-PCR analysis. Overall, the results of these analyses conspicuously showed that the majority of the Hsp families displayed high expression levels at 30 min in all of the studied watermelon tissues . This is thought to be the first reaction against stress. The level of expression then generally decreased at the first hour and increased again during the second hour of stress. It is thought that the Hsp gene increase at 30 min was not enough, and thus it was augmented again in the second hour. In a study on Arabidopsis, it was observed that the expression levels of some Hsp70 genes were upregulated 2–20 times as from 30 min under 40 °C heat stress . In another study based on profiles of Arabidopsis heat shock proteins (Hsp) and transcription factors (Hsf), Hsf and Hsp expression was strongly induced by heat, cold, salt, and osmotic stress, while other types of stress including drought, genotoxic stress, ultraviolet light, oxidative stress, wounding, and pathogen infection induced family or tissue-specific response patterns . In rice, it was detected that some sHsp transcripts started to increase in a short time as 5 min under 41 °C heat stress . When considering the expressions of genes orthologousto ClaHsp genes in poplar, the PtHsp70-09 gene with the number of Potri.003G006300.1, which was orthologous to ClaHsp70-07, and PtHsp90-12 gene with the number of Potri.017G146600.1, which was orthologous to ClaHsp90-04, displayed an increase pattern after salt stress application in various poplar clones . In addition, considering RNA sequence data by Tang et al., PtHsp70-10 with the number of Potri.004G224400.1 and PtHsp70-20 with the number of Potri.009G079700.1, which were orthologous of ClaHsp70-07, were induced under drought stress conditions in the leaf tissues of poplar (Populus trichocarpa) .It can be concluded that the ClaHsp70-07 gene orthologous in poplar has a response against drought and salt stress conditions. ClaHsp90-04 had orthologous genes in Arabidopsis with the numbers of AT5G52640, AT5G56000, and AT5G56010 which have been reported to be heat- and drought-responsive genes in this plant. In addition,AT5G09590, which was orthologous to the ClaHsp70-07 gene, was related with drought and heat reaction in Arabidopsis . According to our study, ClaHsp70-07 genes may be the first response genes to the stress in the stem, root, and tendril tissues of watermelon under combined drought and heat stresses.In addition, the ClaHsp100-67 gene displayed a rapid increase in the first 30 min of stress application in all of the studied tissues of watermelon, and thus may be considered an early response gene under combined drought and temperature stresses. It is understood that the increase in expression levels of the Hsp families in our study is consistent with those studies based on stress responses. It has been determined that there is a relationship between these Hsp genes in watermelon and drought–temperature stress according to the gene expression results. This relationship was also supported by bioinformatics analyses. Considering the changes in expression levels, it is thought that the functions of the mentioned genes can be understood better. In addition, combined drought and temperature stresses were applied and expression levels of The current study provides a detailed genomewide determination of Hsp family members in the watermelon genome, which can help resolve the functions of Hsps. Considering the results and the increase of climate change in the world, this study may be the first step towards the development of high-tolerance watermelon plants, whose products are among the 5 most consumed fresh fruits around the world, against future drought and temperature stresses. In addition, the study contains important literature information for other studies to be done in the future.AcknowledgmentsThis work was completely supported by the Kastamonu University Scientific Research Project Coordinatorship with the project code of KUBAP-01/2016-45.