The aim of this study was to assess the efficacy and safety of laparoscopic holmium laser lithotripsy (LHLL) in the treatment of complicated biliary calculus.We systematically searched the electronic database  up to May 2018 to identify case-controlled studies that compared LHLL with laparoscopic bile duct exploration (LBDE) for complicated biliary calculus.P < .001) and lower estimated blood loss (EBL) , lesser duration of hospitalization  and lower rate of residual stone . There was no statistically significant differences in bile leakage  and hemobilia .Five case-controlled studies were included, with 541 patients (273 in the LHLL group and 268 in the LBDE group). Compared with LBDE, LHLL was associated with shorter operative time (weighted mean difference [WMD] = -40.04, Current evidence suggests that the efficacy of LHLL is superior to that of LBDE but they are similarly safe for the treatment of complicated biliary calculus. Limited by the quantity and quality of the studies included, these conclusions need to be verified by more high-quality studies. The CBDSs are present in about 10–20% of individuals with symptomatic cholecystolithiasis. However, the treatment methods of bile duct stones are varied, for example, open/minimally invasive surgery or radiological methods. It must be noted, however, that there are many methods to manage cholelithiasis, but so far, no single method has shown significant advantages over any other method. There is also no clear treatment for complicated biliary calculus, which is impacted and large, especially in laparoscopic management. The main difficulty is that increases the risk of biliary bleeding, bile duct injury, or bile duct stenosis, lengthening the surgical incision.Laparoscopic cholecystectomy (LC) has become the 1st choice in the treatment of cholecystolithiasis. the 1st to investigate the use of holmium laser together with laparoscopic bile duct exploration (LBDE) in the treatment of CBDS, showed that complete stone clearance from the CBD was achieved in all patients without any postoperative complications. A recent study also revealed that stones were completely removed in 8 patients with complex CBDS by 1-stage laparoscopic holmium laser treatment, and that no postoperative complications developed. Similarly, Xia et al also reported that the application of LHLL improved the success rate of LBDE from 63.5% to 93.7%. To sum up, because the optimal laparoscopic management of complex cholelithiasis remains unclear, our study aimed to explore the efficacy and safety of the choledochoscope and LHLL in the treatment of complex cholelithiasis.Recent studies have reported that laparoscopic holmium laser lithotripsy (LHLL) has good results in impacted CBDS fragmentation in short series. The study conducted by Varban et al2This study is a systematic review and meta-analysis of previously published studies and does not require ethical approval and patient consent.This study was conducted and reported based on the Preferred Reporting Items for Systematic Reviews and Meta-analysis.2.1We searched the relevant publications in the following electronic databases: PubMed, Embase and Cochrane Library, Web of Science, and the Chinese Biomedical Literature Database from January 1966 to May 2018. The following search/Medical Subject Headings (MeSH) terms were used: “bile duct [MeSH] OR bile vessel OR biliary∗ duct OR Common Bile Duct [MeSH] OR choledoch∗ OR Hepatic Duct [MeSH] OR Common Hepatic Duct” AND “calculi [MeSH] stone OR calculus∗ OR lithiasis OR concretion” AND “holmium laser”. In order to search comprehensively, there were no restrictions on the surgical approach or language used in the study, and references lists were also manually reviewed from selected studies. The last search date was May 25, 2018.2.22.2.1The inclusion criteria of this study were based on a “Population, Intervention, Comparison, Outcome, Study” strategy: population, refractory gallstone; Intervention, LHLL; comparison, LBDE; and study, prospective and retrospective observational studies, randomized controlled trial (RCT), case-controlled studies (CCSs), and cohort study (CS).2.2.2The exclusion criteria were as follows: reviews, letters, case reports, and conference abstracts; unavailable full text, unavailable data of our interest, literature with the same author, and Newcastle–Ottawa scale score (NOS) < 6.2.3Outcomes of interest in which the efficacy of the 2 techniques was compared were as follows: intraoperative parameters ; postoperative parameters ; complications .2.4Two researchers (J.P. and J.W.) who had undergone strict evidence-based medical training independently extracted data, such as the name of the 1st author, year of publication, study design, surgical approach, number of participants, age, sex, stone feature and outcomes of interest. Moreover disagreements were resolved by discussion and consensus. The quality of observational studies was assessed by the modified NOS.2.5P-value less than .05 was considered statistically significant.All statistical analyses were conducted by the Review Manager (RevMan) software, version 5.3. . Dichotomous variables were expressed as odds ratio (OR), continuous variables were pooled using weighted mean difference (WMD) with 95% confidence intervals (CIs), and a ,11 In accordance with Higgins’ I2 statistic, heterogeneities < 25%, 25%∼50%, and > 50% were defined as low, moderate, and high, respectively. A fixed effects model was used to calculate the pooled ORs and WMDs when the heterogeneity was low and moderate; otherwise, a random effects model was used for studies with high heterogeneity. When there was high heterogeneity, we used sensitivity analysis to find its source.Statistical heterogeneity among the meta-analysis was tested using the Chi-squared test.33.1A total of 973 related studies were initially obtained in the preliminary literature search. We reviewed 710 results after excluding duplicates by EndNote 6. We finally identified 5 studies that compared LBDE and LHLL in patients with complex biliary calculus, which were gradually and carefully selected by reviewing titles and abstracts and their full text. The literature screening process and results are shown in Figure 3.2Five CCSs were included, with 541 patients (273 in the LHLL group and 268 in the LBDE group). The basic characteristics of the included studies and risk assessment results of bias are shown in Table 3.33.3.1–17 The meta-analysis results of the random effects model showed that the average operative time of 40 min was shorter in the LHLL group and the difference between the 2 groups was statistically significant , and there was a high heterogeneity among the studies  , with a high heterogeneity  , and the heterogeneity was high among studies   . Moreover, total hospitalization cost was also reported in only 1 study, which revealed that the average cost of 8395.22 CNY was greater in the LHLL group and the difference between the 2 groups was statistically significant (P < .05).Time to bowel function recovery was reported in only one study,3.53.5.1,14,16,17 Furthermore, pooled data showed that bile leakage was not significantly different between the 2 groups    by excluding different studies individually. Moreover, the results were not changed, which indicated that these outcomes were robust.4,19 Currently, there are different lasers used for stone fragmentation: holmium: YAG, thulium: YAG lasers, KTP: YAG, and LBO, YAG and diode lasers. However, LHLL has greater flexibility, which increases the access to previously unreachable areas, and its visible diode can help target stones and reduce collateral damage.,22 The good outcomes obtained with the use of lasers in urinary stones prompted their adoption in the treatment of complex cholelithiasis. This study only explored LHLL in the treatment of complex gallstones.Laser lithotripsy has the advantages of high success and low complication rate in the treatment of ureteral stones.The results of the meta-analysis showed that the difference in operative time, EBL, duration of hospitalization, residual stone rate between the 2 operation methods were statistically significant. Compared with those in the LBDE group, the operation time was shorter, EBL was lesser and residual stone rate was lower in the LHLL group. However, there was no statistical difference in bile leakage and hemobilia. In addition, infection of the biliary tract, wound infection, liver function injury, and stricture of the bile duct were reported in only 1 study, and quantitative synthetic analysis was not performed. explored the safety and efficacy of the electronic choledochoscope combined with LHLL in the treatment of complicated cholelithiasis. The results showed that the mean operative time was 67.8 ± 24.8 min, which was similar to the results of this study. Additionally, there were also some findings that are inconsistent with our study. Jun et al showed that the mean operative time was 123 ± 18 min, ranging from 72 to 155 min. The median operative time was 225 min reported by Petersson et al. Inconsistent results may be due to the difference in the skill of the surgeons or different LHLL approaches, through the gallbladder duct or choledochal incision.Operative time, EBL, and duration of hospitalization are important outcome indicators of surgery. This study showed that the mean operative time in the LHLL group was 40 min, which was significantly shorter than that in the LBDE group. Ni et al showed that the average EBL was 26.2 mL in the LHLL group. Although the results were inconsistent with those in our study, the difference was not of clinical significance. The mean duration of hospitalization in the LHLL group was 4 days, and similar results were also validated in this study .The mean EBL in the LHLL group was 56 mL, which was significantly lesser than that in LBDE group. A meta-analysis,25,27 And some studies have shown that in patients in whom clearance of CBDS has been unsuccessful , cholangioscopy-guided holmium laser lithotripsy using endoscopic procedures results in very high stone clearance rates (73–97%).–30 Therefore, the safety of the two surgical methods is similar.Residual stone rate, bile leakage, and hemobilia are the most important outcome indicators in laparoscopic treatment of cholelithiasis. A meta-analysis showed that, compared with LBDE, LHLL could effectively reduce the residual stone rate. In addition, the meta-analysis results also revealed that LHLL could reduce the incidence of major complications , although there was no statistical difference. Similar results can also be obtained from some case analyses. The quality of the methodology of the included study was generally low, mainly because there was no RCT for the comparison of the 2 procedures and only retrospective case-control studies were available, and all of the studies were from China, which inevitably led to selective, implementation, and measurement biases.  Most of the studies did not adequately report complications, which might lead to selective outcome reporting bias.  There was significant heterogeneity in some outcome indicators and the causes might be the difference in the type of holmium laser instrument, size and quantity of stone, etc.  The difference in the surgeon's operative technique, proficiency in the use of machines, and auxiliary staff level will inevitably influence the result. However, the present study data are not sufficient to conduct a subgroup analysis, and more research is needed to provide data support.Limitations of this study: 5Current evidence suggests that the efficacy of LHLL is superior to that of LBDE but they are similarly safe for the treatment of complicated biliary calculus. Limited by the quantity and quality of the studies included, these conclusions need to be verified by more high-quality studies.Conception and design of the study: Tiankang Guo and Yiping Li. Studies selection: Penghui Jin, Wutang Jing and Jia Yang. Data extraction: Caiwen Han, Yuntao Ma and Moubo Si. Statistical analyses: Penghui Jin and Wutang Jing. Wrote the paper: Penghui Jin and Yuanhui Gu. The paper was revised and approved by Penghui Jin.Conceptualization: Weipeng Zhan.Data curation: Weipeng Zhan, Jia Yang, Yuntao Ma.Formal analysis: Penghui Jin, Wutang Jing, Weipeng Zhan.Investigation: Caiwen Han, Moubo Si, Yuanhui Gu.Methodology: Penghui Jin, Wutang Jing.Project administration: Jia Yang, Yuntao Ma.Resources: Caiwen Han, Moubo Si, Yuanhui Gu.Software: Penghui Jin, Wutang Jing.Supervision: Yiping Li, tiankang guo.Validation: Yiping Li, tiankang guo.Writing – original draft: Penghui Jin.Writing – review & editing: Yiping Li, tiankang guo.


Terbinafine has proved to treat numerous fungal infections, including onychomycosis, successfully. Due to its liver metabolization and dependency on the cytochrome P450 enzyme complex, undesirable drug interaction are highly probable. Additionally to drug interactions, the treatment is long, rising the chances of the appearance of side effects and abandonment. Pharmacokinetic data suggest that terbinafine maintains a fungicidal effect within the nail up to 30 weeks after its last administration, which has aroused the possibility of a pulse therapy to reduce the side effects while treating onychomycosis. This study’s goal was to evaluate the effectiveness of three different oral terbinafine regimens in treating onychomycosis due to dermatophytes. Sixty-three patients with onychomycosis were sorted by convenience in three different groups. Patients from group 1 received the conventional terbinafine dose (250 mg per day for 3 months). Group 2 received a monthly week-long pulse-therapy dose  and group 3 received a 500 mg/day dose for 7 days every 3 months, totaling four treatments. There were no statistical differences regarding the effectiveness or side effects between the groups. Conclusion: A quarterly terbinafine pulse regimen can be a possible alternative for treating onychomycosis caused by dermatophytes. Onychomycosis due to dermatophytes, yeasts, and non-dermatophyte molds comprises 50% of all cases of nail disease . Known iThe susceptibility to onychomycosis is inherited and it is often observed among family members. Some studies revealed the existence of polymorphisms in genes of the major histocompatibility complex related to higher susceptibility to onychomycosis from dermatophytes, particularly haplotypes HLA-DR8 and HLA-DR1 ,10,11Trichophyton rubrum, Trichophyton mentagrophytes, Epidermophyton floccosum, and Microsporum spp. The remaining infections can be due non-dermatophyte molds, predominantly Scopulariopsis brevicaulis, Aspergillus spp., Acremonium, Fusarium sp.p, Alternaria alternata, and Neoscytalidium spp., or to yeasts, such as Candida albicans [The estimated global prevalence of onychomycosis is 5.5% . More thalbicans .Trauma caused by shoes produces toenail changes, especially in people with orthopedic changes that cause faulty poor adaptation of the feet in shoes, that are identical to some onychomycosis at the time of clinical presentation . Most toAside from the high prevalence, onychomycosis has therapeutic challenges. The available therapeutic arsenal is not vast, and there are high rates of resistance and recurrence, making it a noteworthy public health issue ,17.Topical and oral antifungals are the treatment options for onychomycosis. Topical therapy is used in children and adults with mild to moderate onychomycosis or for single affected digits. Ciclopirox and amorolfine are the most used topical agents, and recently, tavaborole and efinaconazole have been introduced in North America ,19,20.FDA-approved oral treatment for onychomycosis includes terbinafine and itraconazole, and fluconazole is used off-label. Due to fewer collateral effects and higher cure rates, terbinafine is usually preferred over itraconazole . The staThe standard dose for a terbinafine pulse regimen is 500 mg per day for 7 days a month, twice or three times for fingernails and three to four times for toenails .T. rubrum.Zaias and Rebell  have desWe performed an open non-randomized study in which standard terbinafine regimens were compared with a pulse terbinafine regimen of 500 mg/day for 7 days every 3 months to treat onychomycosis caused by dermatophytes.In total, we included 63 patients  aged between 24 and 70 years who had visited the Dermatology Outpatient’s Clinic at Santa Casa de Curitiba Hospital between August 2013 and July 2016. Those aged 18 years or older and diagnosed with dermatophyte onychomycosis based on clinical manifestations and confirmed using mycological culture were eligible to participate. Patients with less than 25% of the nail affected by the disease, liver or kidney impairment, pregnant or lactating were excluded. All participants signed an informed consent term.Two measurements were taken of infected nails before, during the appointments, and at the end of each group treatment. The first one was the length of the nail plate from the free edge to the proximal nail fold, and the second one was the length of the visible fungal-infected portion. The percentage of the compromised nail was then calculated.Data concerning age, sex, occupation, sport activities, comorbidities, and concomitant use of medications were recorded. Patients were divided into three groups, according to their order of attendance.In Group 1, 20 patients received continuous 250 mg terbinafine for 3 months. In Group 2, 21 patients received a terbinafine 500 mg monthly pulse regimen, for 4 months. In Group 3, 22 patients received terbinafine 500 mg/day for 7 days, every 3 months and completed four pulse regimens.Patients of Group 1 were asked to attend the hospital monthly. Patients of Group 2 had appointments every 2 months. Patients of Group 3 attended the hospital every 3 months. During the appointments, all patients were examined, questioned concerning any possible side effects, and received a new supply of terbinafine containing 28 tablets.Mycological cultures were provided for all patients who completed the study.The degrees of improvement were classified as follows: total cure (TC), clinical disease-free nail and a negative mycological culture; mycological cure (MC), <25% of nail impairment and a negative mycological culture; clinical improvement (CI), <25% of nail impairment and a positive mycological culture; therapeutic failure (TF), unchanged clinical examination or worsening and a positive mycological culture.p value of 5% was considered statistically significant.For the quantitative variables, a comparison between treatments was undertaken using a Kruskal–Wallis non-parametric test, suitable for the analysis of independent samples and variables with interval measurements without normal distribution . For theTrichophyton sp, T. rubrum, T. mentagrophytes, and Microsporum gypseum  in all three groups, followed by the 4th, 5th, and 3rd toenail and the thumbnail (n = 20). There was no significant difference among the groups regarding the distribution of affected fingers or toenails , followed by DLSO with subungual hypertrophy (n = 25), total dystrophic onychomycosis (TDO) (n = 7), proximal subungual onychomycosis (PSO) (n = 3), and superficial onychomycosis (SO) with deep invasion (n = 1). Nine patients had more than one nail type of onychomycosis in different nails; therefore, for the clinical classification, the sample included 71 affected nails , 10 (71.43%), and 13 (86.67%) patients from groups 1, 2, and 3, respectively, presented with TC, MC, or CI .p = 0.280) concerning the response to terbinafine. However, among the patients who finished the study, the majority  showed at least one degree of improvement .After applying the chi-square test, no significant difference was observed between the groups  of them because of side effects. The other 15 (23.90%) patients dropped out for personal reasons.n = 4, Group 3, n = 1) and cutaneous rash . None of these patients had comorbidities or used medications that could interact with terbinafine.  in previous studies ,49 and arubrum, in our study, two patients of Group 3 (13.33%) had TC and one patient (6.66%) had clinical improvement of onychomycosis caused by T. mentagrophytes. We opted for the 500 mg quarterly dose in order to compare the efficacy with that of the standard 500 mg monthly dose used in most of the published studies based on pulse regimens. In addition, this is the terbinafine dose of pulse regimen utilized in Brazil. Further studies with a more significant number of participants are necessary to compare the effectiveness of the trimester schemes of oral terbinafine in the treatment of all types of dermatophyte onychomycosis.Compared to the Zaias and Rebell’s study , which dTreatment outcomes for onychomycosis can also vary according to age, clinical presentation, comorbidities, and the use of medications that may interact with antifungal treatments ,51,52. DThe asymmetric gait nail unit syndrome (AGNUS), firstly described by Zaias et al. in 2012, is caused by repetitive toe trauma in a closed shoe in patients with asymmetric walking due to orthopedic abnormalities. The resulting nail changes can undoubtedly be clinically identical to onychomycosis . In our n = 7), obesity (n = 5), hypothyroidism (n = 3), diabetes (n = 1), and HIV active infection (n = 1). Only one patient with a comorbidity, who had hypothyroidism, presented with TF.As life expectancy is increasing globally, it is common for patients with onychomycosis to present with comorbidities and take more than one long-term medication ,57,58,59The pulse regimen therapy (groups 2 and 3) had the lowest cost of treatment. Group 1 used a total of 84 tablets, while groups 2 and 3 used 56 tablets each. The quarterly pulse regimen had the same cost compared to the conventional terbinafine pulse regimen but might be more financially appealing since patients are required to purchase a second terbinafine supply only six months after the start of treatment ,62,63.The long drug rest interval may compromise adherence to treatment due to patients forgetting the quarterly dose, which is an issue that can be addressed through counseling patients, family members responsible for them, or, eventually, caregivers of elderly persons using a simple reminder message recorded on their cell phone .Despite the limited sample, a pulse therapy regimen using terbinafine 500 mg per day for a week every three months was found to be a potentially useful alternative in the treatment of onychomycosis by dermatophytes. Further studies involving a more significant number of patients are necessary to confirm the effectiveness of this treatment regimen.


Children are in the risk group for developing hypovitaminosis D. Several strategies are used to reduce this risk. Among these, fortification of foods with vitamin D (25(OH)D) has contributed to the achievement of nutritional needs. This systematic review aims to discuss food fortification as a strategy for maintenance or recovery of nutritional status related to vitamin D in children. The work was developed according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and registered in the International prospective register of systematic reviews (PROSPERO) database (CRD42018052974). Randomized clinical trials with children up to 11 years old, who were offered vitamin D-fortified foods, and who presented 25(OH)D concentrations were used as eligibility criteria. After the selection stages, five studies were included, totaling 792 children of both sexes and aged between two and 11 years. Interventions offered 300–880 IU of vitamin D per day, for a period of 1.6–9 months, using fortified dairy products. In four of the five studies, there was an increase in the serum concentrations of 25(OH)D with the consumption of these foods; additionally, most children reached or maintained sufficiency status. Moreover, the consumption of vitamin D-fortified foods proved to be safe, with no concentrations of 25(OH)D > 250 nmol/L. Based on the above, the fortification of foods with vitamin D can help maintain or recover the nutritional status of this vitamin in children aged 2–11 years. However, it is necessary to perform additional randomized clinical trials in order to establish optimal doses of fortification, according to the peculiarities of each region.  Vitamin D (25(OH)D) is an important nutrient during childhood because of its involvement in bone formation, as well as in the immune system, which can result in higher body needs for this vitamin ,2,3. ThuDietary intake is one of the ways to obtain this vitamin, but the food contribution is limited (10% to 20%) ,5,6. TheEven in sunny countries, vitamin D deficiency is observed in different population groups ,11,12,13There are few clinical trials evaluating food fortification as a strategy to improve or maintain vitamin D nutritional status in child population ,17,18,19This study was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement (PRISMA)  Supplem and suppFor the construction of this review randomized clinical trials (RCTs) with children up to 11 years old, who presented data regarding serum or plasma vitamin D (25(OH)D) concentrations at baseline and after the intervention, were considered eligible. The comparators were foods fortified with cholecalciferol or ergocalciferol with non-fortified foods.Animal or in vitro studies, manuscript published only in summary form, and review studies were excluded. In addition, studies including children diagnosed with diseases that compromised vitamin D metabolism were excluded. To avoid interference from other nutrients, studies who offered fortified foods with vitamin D in conjunction with other nutrients were not considered.ClinicalTrials.gov from inception to January 2019. A gray-literature search included Google Scholar and OpenThesis. The structured search strategy used the following terms: (child) AND   . The seaTwo evaluators (B.C.S and P.N.B-L) conducted all the selection stages of studies independently, first reading the Titles and Abstracts, and subsequently reading the full studies selected in the first stage. Any disagreement was resolved in conjunction by three evaluators .To evaluate the agreement between the evaluators in the selection stages of the studies, the kappa coefficient proposed by Landis and Koch  was usedThe following information was extracted from studies: characteristics of the participants; season of the year; dosage and food matrix used as the fortification vehicle; sun exposure; duration of intervention; and 25(OH)D status. The cut-off points proposed by the World Health Organization were adopted for the classification of BMI (Body Mass Index) for age, with z-score values ≥ −2 and ≤+1 considered normal weight .In the included study that presented the serum 25(OH)D concentration as a graph , mean anThe risk of bias in the studies was independently assessed by two reviewers using the Cochrane Collaboration’s tool for assessing risk of bias in randomized trials . The tooIn order to assess the main outcome, vitamin D intake values were converted into international units per day µg × 40 = IU) [0 = IU 4. HoweverThe graphical representation of the bias risk analysis was elaborated with Review Manager software 5.3 .The initial search identified 1778 studies, and, after all the selection stages, five studies met the inclusion criteria and were included in this review ,17,18,19Of the included randomized clinical trials, four are double-blind type ,16,17,19Regarding the analysis of bias risk , among tThe evaluation of detection, attrition, and reporting bias showed that 100% of the studies presented low risk of bias ,16,17,19According to the analysis of other sources of bias (assessment of sun exposure and skin color), 60% of the studies presented low risk ,17,19. On = 568) and control (n = 224). Regarding nutritional status, three studies were conducted with normal weight children according to BMI for age [The five studies selected present data on 792 children of both sexes and aged between two and 11 years, distributed in the intervention groups with vitamin D-fortified foods  ,19, and Another relevant aspect to be evaluated is sun exposure. In two studies, no significant differences were observed regarding direct sun exposure among the evaluated children ,17. The The children’s vitamin D status at baseline was variable, with the presence of children at risk of deficiency (<30 nmol/L) and of insufficiency (30–49 nmol/L), and with status classified as sufficiency (≥50 nmol/L) of vitamin D . HoweverCorroborating aspects of vitamin D status, four studies also considered the usual intake of this vitamin. Different methods of assessing food intake were used; among them, semiquantitative food frequency questionnaire (FFQ) , short FThe results showed that at baseline, dietary vitamin D intake was lower than the adequacy recommendations adopted in each study. Dairy products used as vehicles for vitamin D fortification were cheddar cheese, yogurt ,17, and By observing the effects of intake of fortified foods on serum vitamin D levels, in three studies, an increase of 25(OH)D levels after the intervention period was observed in all groups receiving fortified foods, whereas the respective control groups did not present alteration of this vitamin in the serum ,17,19 T.In the study by Hower et al. , the forDifferent behavior was observed in the study by Brett et al. , in whicDifferently, Ohlund et al.  observedThe methods used to determine serum 25(OH)D concentrations were liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) ,19, highIn none of the studies, serum 25(OH)D concentrations were above safety limits (>250 nmol/L) in the groups receiving fortified foods, even during the months with abundant sunshine.This study evaluated the available evidence regarding food fortification as a strategy for maintenance or recovery of vitamin D nutritional status in children. The results of the individual studies suggest that consumption of vitamin D-fortified foods seems to be an important strategy to reduce the prevalence of hypovitaminosis D in this age group, which present greater vulnerability to developing this deficiency. However, due to the clinical and methodological heterogeneity of these studies, a meta-analysis could not be performed.Previous systematic reviews and meta-analysis sought to evaluate the effect of fortification of foods with vitamin D, demonstrating a significant increase in 25(OH)D concentrations from the consumption of these foods in different population groups ,20,21,29The effects of fortification with vitamin D are reported to be dependent on increased intake of fortified foods, previous vitamin D status, age, body composition, skin pigmentation, sun exposure, geographic aspects, and food intake ,30,31,32As noted in the included studies, the use of fortified foods to improve or maintain vitamin D status considered the consumption of milk and dairy products as part of the population’s eating habits. Milk and its products are among the foods often fortified with vitamin D, as they are widely consumed by children, especially in developed countries, and have good bioavailability of the vitamin ,34.Dairy products naturally feature in their composition nutrients that act together with vitamin D, thus contributing to the metabolism of this vitamin, as well as in the physiological processes in which it is active .Furthermore, vitamin D is directly related to the calcium present in these foods, in which the 1,25(OH)2D stimulates the intestinal absorption and renal reabsorption of this mineral, as well as acting in the processes of bone mineralization, due its participation in the synthesis of osteocalcin . At the In the countries of origin of the included clinical trials, in areas with restricted sun exposure, it is noted that, although increasing, food fortification is still not widely adopted. In Canada, where two of the five included studies were conducted ,17, onlyDifferent responses to food fortification at serum vitamin D concentrations can be observed depending on previous nutritional status ,18. ChilWhen circulating 25(OH)D concentrations are present in sufficiency, a portion of this vitamin can be transferred to body stores once saturation of the vitamin D 25-hydroxylase (CYP2R1)—the enzyme responsible for vitamin D hydroxylation in the liver—occurs ,17. The n = 7; 6.9 nmol/L; 95% CI: 3.7, 10.0 nmol/L; I2 = 99.9%) than trials that offered daily supplements  (p = 0.001), and bolus injections  (p = 0.04). The authors demonstrated that the serum 25(OH)D response to vitamin D intake differed on the basis of baseline status, intakes, and delivery mode, but not age, sex, or latitude [The discussion of dose-response of fortification at 25(OH)D concentrations is little explored. Brett et al.  observedlatitude . HoweverI2 = 89%) was observed in the 25(OH)D concentrations for each 40 IU day of vitamin D ingested from fortified foods. In the paper by O’Donnell et al. [n = 466 individuals; 138–800 IU vitamin D/day) also demonstrated an increase in the 25(OH)D concentrations in adults  nmol/L; I2 = 0.0%; p = 0.77).A meta-analysis published by Black et al. , performl et al. , the comFrom the evaluated results, it is not possible to determine the optimal fortification dose, since changes in 25(OH)D concentrations were observed in almost all trials in different populations. However, the dose range of vitamin D used in fortification of foods from included studies (300–800 IU/day) were effective in maintaining or recovering vitamin D status in children. Besides, the optimal dose could be dependent on the characteristics of the population targeted, including those with vitamin D deficiency.The intervention period, another relevant aspect for the study outcome, varied among the studies. In some of the included studies, this variable was chosen because it corresponds to the length of the season of interest. Even so, all studies exceeded the considered half-life of 25(OH)D which is between 2 and 3 weeks . VitaminIn children, deficient vitamin D status is the main cause of rickets, a disorder characterized by disturbances in bone growth and skeletal abnormalities, besides negatively influencing cognitive development, hormone formation, and immune function ,47,48. IIn addition, other variables that may affect the vitamin D bioavailability should be considered. Body composition is an important aspect for understanding the metabolism of this vitamin because adiposity has been inversely associated with 25(OH)D concentrations by acting on vitamin D storage, increasing its clearance ,17,50,51Overall, studies have shown that fair-skinned individuals have higher levels of 25(OH)D compared to darker skinned individuals ,52, a faGeographical location and climatic characteristics also directly influence the synthesis of vitamin D in the skin, with low or absent synthesis observed during most of the winter in places with latitudes above 33° . In thisIt is known that diet has a lower participation in 25(OH)D concentrations . This isIn addition, methods of serum 25(OH)D assessment have a strong influence on the diagnosis of vitamin D status. There is no consensus regarding the reference standard due to susceptibility to problems in the pre-analytical, analytical, and post-analytical phases. Immunoassays are dependent on the specificity of the antibodies used, not being able to identify the 3-epi-25(OH)D molecule. The HPLC method has been replaced by the LC-MS/MS method, currently considered the gold standard because of its higher sensitivity ,54. It sThis is the first systematic review that addresses the effects of vitamin D-fortified food intake by children aged 2–11 years, taking into account the other factors that influence vitamin D metabolism, such as latitude, sun exposure, skin color, use of sunscreen, and usual dietary intake. Furthermore, this review included studies that offered fortified foods exclusively with vitamin D in order to minimize additional factors that could interfere in the bioavailability of this vitamin.Regarding limitations of the present study, it is possible to mention the low number of studies available in the literature that offered vitamin D-fortified foods for children. Thus, this study recommends further randomized clinical trials of this age group to increase evidence and identify the optimal dose for vitamin D food fortification. Moreover, it should be emphasized that the habitual consumption of the evaluated groups should be considered, even in the face of the limitations inherent in the methods used to evaluate habitual nutrient intake. The results of the individual studies suggest that the fortification of foods with vitamin D can be used for maintaining or recovering the vitamin’s nutritional status in children aged 2–11 years. In addition, the intake of 300–880 IU of vitamin D per day through the consumption of fortified foods appears to be safe under the conditions studied, with no increase in serum 25(OH)D concentrations above the tolerable limits. However, due to the insufficient number and heterogeneity between the studies, a meta-analysis evaluating the outcomes of interest was not performed, and a pragmatic recommendation on the fortification of foods with vitamin D is not possible. It is necessary to carry out additional randomized clinical trials in order to increase the strength of evidence, and to establish ideal doses of fortification at the population level.


A search for the “magic bullet”, a molecule, the targeting abilities of which could stop the migration of tumor cells, is currently underway, but remains in the early stages. There are still many unknowns regarding the cell migration. The main approach is the employment of mouse models, that are sources of valuable information, but still cannot answer all of the questions. One of the molecules of interest is Transglutaminase 2 (TG2). It is a well-described molecule involved in numerous pathways and elevated in metastatic tumors. The question remains whether mice and humans can give the same answer considering TG2. Tumors consist of a heterogeneous population of cells, and the heterogeneity is the consequence of genomic instabilities such as chromosomal rearrangements, DNA mutations, and epigenetic changes . MalignaThere are two main forms of cell migration: single cell and collective cell migration. Recently, it has been suggested that the migration of cancer cells is more plastic, thus tumor cells can change their methods of movement from one form to another, depending on external signals and intracellular regulation . A develAt the biochemical level, EMT depends on signaling pathways, leading to the up-regulation and down-regulation of biomarkers responsible for the phenotype of mesenchymal and epithelial cells by the transcription factors Snail, Twist, Slug/Snail2, Zeb1, and Zeb2/SIP1 ,15,16. EThe migration can be considered collective if cells migrating keep contact and influence one another while moving . The colDictyostelium, which is characteristic of membrane blebs and has no need for MMPs [On the other hand, in the invasive front of melanoma, TGFβ influences amoeboid migration, since it promotes cell rounding, membrane blebbing, contractility and invasion . Melanomfor MMPs . The maifor MMPs . One of for MMPs . For exafor MMPs . Additiofor MMPs . It is nfor MMPs ,33.Epithelial–mesenchymal transition in mouse tumorigenesis was described for the first time over 100 years ago, using the term “carcinosarcoma” ,35. EMT In human tumors, EMT has been associated with metastasis, drug resistance, and tumor recurrence . For exaThere is a possibility that the EMT-phenotype may be misleading as a marker for metastases in murine models. The 67NR cells show features of EMT, including the spindle phenotype and a loss of E-cadherin, but they are non-metastatic in vivo, which could be proof that the spindle phenotype is a biological dead-end for mice. In support of this finding, highly metastatic mouse breast cancer 4T1 cells are epithelial-like with all of the characteristics of epithelial cells, such as the presence of E-cadherin and the lack of N-cadherin. On the contrary, the metastatic 66cl4 cell line isolated from the same type of tumor as the 4T1 cells and characterized with high expression of N-cadherin and the loss of E-cadherin is almost non-migratory and does not express invasive properties in vitro. All of the above factors suggest that migratory and invasive properties in vitro are not connected with EMT markers and metastatic potential in vivo for the mouse breast cancer models . Besides2+ is a significant regulator of cell migration, and transitory Ca2+ release from the intracellular depos is necessary to induce membrane blebs in D54 glioma cells isolated from BALB/c Scid mice in situ. Bleb formation can be disturbed by affecting intracellular Ca2+ homeostasis [Intravital imaging of B16F2 melanoma cell migration in mice showed the heterogenous behavior of the cells. Some melanoma cells migrated as single cells, while others followed. Single cells used an amoeboid type of motility, while the cells following the same path used multicellular streaming . Additioeostasis . Moreoveeostasis .2+ (Transglutaminases (TGs) are the family of enzymes that catalyze covalent bond formation between free amino groups  and the γ-carboxamide groups of peptide-bound glutamine . In 19572+ , and tisTissue transglutaminase is present in the cell’s cytosol, mitochondria, and nucleus, and it can be found on the cell surface and in the ECM with a ubiquitous presence throughout the body ,55. BesiActivated tissue transglutaminase introduces intermolecular isopeptide bonds and thus modifies ECM proteins. The bonds have an influence on the stability of the extracellular matrix and cell adhesion/spreading. Substrates of TG2 include fibronectin, collagen, fibrinogen, fibrin, laminin/nidogen, osteopontin, and vitronectin . These bElevated expression of TG2 has been found in secondary metastatic tumors of glioblastoma , pancreaTransglutaminase 2 on the cell surface is involved in cell adhesion via its role as an integrin-binding coreceptor for fibronectin and a stabilizer of ECM ,79. Cellhigh/CD24-/low stem cells markers, while influences down-regulation of an epithelial marker CD326 [+/CD117+ stem cell markers is significantly reduced by the knock-down of TG2 and proposed mechanism is dependent on TGFβ signaling [In secondary metastatic and chemotherapy resistant tumors, TG2 is often up-regulated, whereas during tumor development and in primary tumors, TG2 is down-regulated . The decer CD326 . Similarignaling .−/− mice have normal collagen crosslinking and are not protected from liver fibrosis, suggesting that the lysyl oxidase (LOX) family has a role in disease progression [−/− mice have shown that there is no role for TG2 in glucose homeostasis, at least in mice [−/− mice have fragmented mitochondria with altered morphology, depolarization of the mitochondrial membrane, and reduced reactive oxygen species (ROS) formation [The crosslinking activity of TG2 influences ECM deposition and angiogenesis in mouse models, as shown after intratumor injection of active TG2 into mice with CT26 colon carcinoma tumors . Furthergression . Althoug in mice , in diab in mice , thus sh in mice ,96, and ormation ,97,98. Mormation ,100.2+. Mitochondrial Ca2+ homeostasis is of great importance for the migration of cells [2+ homeostasis, such as the voltage-dependent anion-selective channel (VDAC) protein, prohibitin, and different ATP synthase subunits, were discovered to be substrates of TG2 [The main question that we need to consider is whether mouse models can mimic all aspects of a human disease. The physiological differences between mice and human are numerous, including differences in lifespan, structure of the stromal elements , and theof cells . After ss of TG2 ,112, ind


To examine the efficacy and safety of the toothpaste containing Rhizoma Chuanxiong and Rhizoma Imperatae extracts in reducing gingivitis.N = 60) or the control group (N = 60). Tetramethylpyrazine, senkyunolide A, ferulic acid, and ligustilide are the main effective components of Rhizoma Chuanxiong and Rhizoma Imperatae contains the main components of cylindrin, carotene, 5-hydroxytryptamine, potassium, and calcium. The control group used placebo toothpaste containing neither Rhizoma Chuanxiong extract nor Rhizoma Imperatae extract. Plaque, gingivitis, and bleeding were assessed at the baseline, prior to the supragingival scaling, and at 4, 8, and 12 weeks. A double-blind clinical trial was conducted, in which 120 volunteers were randomly assigned to the test group . After 4 weeks of usage, there were no statistically significant differences in all of GI, BI, and BOP% scores between the two groups. However, the decrease became statistically significant at next two intervals  in the efficiency of GI, BI, and BOP% which was 8.04%, 11.02%, and 37.16%, respectively. There were no treatment-related adverse events reported. During the trial, both test and control groups showed a decreasing trend compared to the baseline. At the end of 12 weeks, with respect to Gingival Index (GI), Bleeding Index (BI), and Bleeding on Probing percentage (BOP%) scores, there were significant differences between test and control groups (GI, The toothpaste containing Rhizoma Chuanxiong and Rhizoma Imperatae extracts was well tolerated and significantly reduced gingivitis and bleeding after usage for 12 weeks. There was better improvement at molars, and the more serious the baseline status was, the better the efficacy was. Chronic gingivitis is one of the most common oral diseases with high prevalence around the world . A surveDental plaque is the major etiological and initiating factor for the development of gingivitis . Therefo Ligusticum chuanxiong Hort, known as a famous medicinal herb from Sichuan, and its main components include volatile oil, acid composition, and nitrogenous compounds 2 [According to the formula n1=n2 = 2[(ZZβ)S/δ]2 , consideZβ)S/δ]2 , 37, 120There were no statistically significant differences neither in age nor in gender between the two groups. There were also no statistically significant differences on PI, GI, BI, or BOP% between the two groups , which d P<0.001, BI, P<0.001, and BOP%, P<0.001, resp.). However, the differences between two groups were not statistically significant with respect to all indexes at the 4-week and 8-week time intervals and the PI score between test and control groups at any time interval  for all groups and all parameters are given in P>0.05) and their variances were also homogeneous (P>0.1). The results of ANOVA showed that there were significant differences between molar and premolar or anterior teeth with respect to GI and BI scores.It was reported that there were significant variations in plaque accumulation within dentition, and tooth position within the alveolar bony strongly correlated with gingival recession , 39. The P=0.010) and BI  scores, revealing that the more serious the baseline status, the better the effect of toothpaste  between the two groups of subjects .P=0.648, P=0.829).No bad/terrible-taste or hard-to-accept was reported in both groups, and the difference of the constituent ratio between two groups was not statistically significant with respect to taste and tolerance degrees . However, the reductions became statistically significant in the next two intervals . The efficiency of GI, BI, and BOP% was 8.04%, 11.02%, and 37.16%, respectively. The results demonstrated that the herbal toothpaste was effective in inhibiting gingivitis and oral health maintenance when compared to the negative control toothpaste. Moreover, a better efficacy was also found in the molars than the premolars and the anterior teeth. The effect has strong correlation with the severity at baseline with respect to GI  and BI  scores; the more serious the baseline status is, the better the effect is. It is indicated that more serious gingivitis may achieve a better improvement when treated with herbal toothpaste.After 4 weeks of use, there were no statistically significant differences in the reduction of all parameters between the two groups (Shen Nong Ben Cao Jing) more than 400 years ago. In traditional Chinese medicine beliefs, Rhizoma Chuanxiong has the effect of invigorating blood and expelling wind-damp as well as relieving pain. During the past years, valuable information has been obtained on its pharmacology. Rhizoma Chuanxiong mainly contains organic acid, essential oils, and nitrogenous compounds. Tetramethylpyrazine, senkyunolide A, ferulic acid, and ligustilide are the main effective components [β, IL-6, and TNF-α and also reduced the expression levels of cyclooxygenase-2 (COX-2) as well as inducible nitric oxide synthase (iNOS) [2O2 [As an authentic herbal medicine of Sichuan, Rhizoma Chuanxiong was first recorded in the Divine Husbandman's Classic of the Materia Medica  , 49, 50.OS) [2O2 . BesidesOS) [2O2 , 53. In OS) [2O2 . The bloOS) [2O2 . Imperata cylindrica (L.) was reported to show antimicrobial activity on Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and Staphylococcus aureus [ Imperata cylindrica (L.) were reported to show the anticancer activity in human oral cancer cell line [Rhizoma Imperatae is also a common Chinese herbal medicine, of which the main components include cylindrin, carotene, 5-hydroxytryptamine, potassium, and calcium. Modern pharmacological research shows that Rhizoma Imperatae mainly has functions of hemostatic, diuresis, antihypertensive, bacteriostasis, anti-inflammatory, analgesic, and antitumor, reducing hydroxyl radical, antioxidant, and enhancing immunity , 27. Thes aureus . The metell line . Isoeugeell line . The polell line . At presell line .Existing theories show that Rhizoma Chuanxiong companied with Rhizoma Imperatae has great application potential and value in the treatment of gingivitis. The efficacy of the variety of Chinese herbal toothpaste has been studied previously. However, there are few standardized clinical studies on Rhizoma Chuanxiong or Rhizoma Imperatae regarding the extract-containing toothpaste at present. The results from the current clinical study could not be directly compared with similar studies. And this is the first study reported in the medical literature which explores the effects of toothpaste containing Rhizoma Chuanxiong and Rhizoma Imperatae extracts on dental plaque and gingivitis. Effects of herbal toothpaste on reducing plaque formation and gingival inflammation were investigated in clinical studies and results were equivocal, which demonstrated significant plaque reductions ranging from 7.17% to 61.2% and gingivitis reductions ranging from 5.20% to 70.6% –61.When the study ended, both types of toothpaste were effective in decreasing plaque and gingivitis parameters compared with the baseline. And the experimental group showed a decreased trend, while the control group went up at the end of 12 weeks. The reduction in indexes in the control group may be attributed to ultrasonic cleaning and repeated oral education, which probably improve oral health a lot. However, this influence was temporary, and the herbal toothpaste can help maintain this improvement. Moreover, the current study showed a better improvement in the molars. And the more serious the baseline status is, the better the effect is. It could be speculated that the molar is not easy to be cleaned resulting in worse health condition, and the herbal toothpaste has a broad application prospect in the treatment of gingivitis, especially severe inflammation. The reduction of dental plaque between test and control groups has no significant difference, while the bleeding improved a lot in test group. These phenomena may be attributed to the anti-inflammatory blood circulation, which improves the functions of Rhizoma Chuanxiong and Rhizoma Imperatae; even no obvious antibacterial effect was observed or reported. All of these may provide the theoretical basis of promoting blood circulation and removing stasis in traditional Chinese medicine. In addition, the observed effect in present study may be attributed to participants' awareness of enrolling in oral hygiene study—Hawthorne effect, no matter what toothpaste they receive. Therefore, extending observation time to a longer period will provide a more powerful comparison.As the placebo-controlled setting can minimize the subjective expectation effect and bias of subjects and researchers and directly measure the difference in efficacy and safety between the test drug and placebo, it can give the appropriate conclusion of the test drug with a smaller sample. For the positive control, there is little difference between the efficacy of the test drug and that of the positive control drug, so a larger sample is needed to achieve the same efficacy in order to detect the difference between the two drugs. It would be better to set both placebo control and positive control, but, considering our study purpose, the test conditions, manpower, and material resources, only placebo control was chosen, which was the limitation of our study. We would add “positive control” group in our similar clinical trials in the future.Within the limits of this clinical study, regular use of the herbal toothpaste containing Rhizoma Chuanxiong and Rhizoma Imperatae extracts could effectively and safely reduce gingivitis of the study subjects. There is a better improvement at molars, and the more serious the baseline status is, the better the effect is. Further long-term studies are needed to confirm its benefits.


BorSal” for this combined boron toxicity and high salinity state in the soil. Membrane-bound activities, mobility of ions, water transport, pH changes, transpiration, photosynthesis, antioxidant activities, and different molecular transporters are involved in the effects of BorSal interaction in plants. The discussed mechanisms indicate that the BorSal stress state should be studied in light of the involved physiological and molecular processes that occur after B and salt interaction in plants.The continuously changing environment has intensified the occurrence of abiotic stress conditions. Individually, boron (B) toxicity and salinity stress are well recognized as severe stress conditions for plants. However, their coexistence in arid and semi-arid agricultural regions has shown ambiguous effects on plant growth and development. Few studies have reported that combined boron toxicity and high salinity stress have more damaging effects on plant growth than individual B and salt stress, while other studies have highlighted less damaging effects of the combined stress. Hence, it is interesting to understand the positive interaction of this combined stress so that it can be effectively employed for the improvement of crops that generally show the negative effects of this combined stress. In this review, we discussed the possible processes that occur in plants in response to this combined stress condition. We highly suggest that the combined B and salinity stress condition should be considered as a novel stress condition by researchers; hence, we recommend the name “ In modern research, the majority of abiotic stress-based responses that are verified in laboratories or controlled conditions occur differently under field conditions . One of Boron (B) and salinity are two drastic individual abiotic stress conditions largely responsible for crop losses ,5. B toxHigh soil salinity affects 20–50% of irrigated agricultural land with an approximate annual economic drop of 12.6 billion USD . HoweverBorSal) so that agricultural solutions for this issue can be pursued by considering the involved mechanisms. To develop a genetic engineering-based amendment, it is extremely crucial to understand the involved mechanisms at the molecular level.In the last few years, attempts have been made to understand the combined effects of these stresses on different crops, but no clear conclusions have been obtained regarding their interactive effects ,24,25,26Because of the continuously changing environment, plants are being increasingly exposed to combinations of different stresses as compared to individual stresses. The simultaneous occurrence of abiotic stresses instead of individual stress condition can be more fatal to crops ,28,29; hPlants show both unique and shared responses to adapt themselves to combined stress conditions . On the B stress affecting crop productivity is often accompanied by soil salinity. Crops in arid or semiarid agricultural areas facing high salinity problems often experience B stress due to limited leaching ,48,49,50Although some studies have been conducted to determine the effect of B and salinity as individual stresses ,57,58,59Few reports have shown negligible effects of combined BorSal stress on the shoot weight of different cultivars ,61,62,632SO4) also causes variations in B accumulation in plants. A number of studies have investigated the interactive effects of NaCl and Na2SO4 salinity under B toxicity and have obtained varying results [2SO4 salinity [Increasing salt concentrations may add to the negative (damaging) effect of B toxicity on plant growth. In wheat, high B and salt concentrations can be more disastrous to plants in the form of combined stress having a suppressive effect on shoot dry weight and grain yield . However results ,69,70,71salinity ,72.Plants acclimatize to this BorSal condition with the help of both “unique” and “shared” stress responses. Few responses are unique to B and salinity stress, while others are common to both the stresses. B absorption, exclusion mechanism, membrane bound activities, and chemical changes in plants are among the important channels involved in reducing B toxicity under saline conditions ,70,73,74The nature of the interaction of salinity and B in plants is not conclusive and needs to be thoroughly understood . RecentlB has significant involvement in maintaining the stability of the plasma membrane, and B-induced changes in the cell may affect ion transport and other membrane-related processes ,77,78 Fa.In plants with the augmentation of B toxicity under saline conditions, changes in the soluble B concentration in different cell compartments need to be discussed. Few studies have reported high B concentration in the apoplast under B toxicity condition , while oIn sunflower, lower B in the apoplasm and higher accumulation of B in the symplasm was observed , and it In wheat, enhanced B accumulation was observed in the apoplasm as compared to that in the symplasm in normal to high B conditions. Consequently, B uptake occurs via passive diffusion through the plasma membrane . In diffSalt supply in the presence of high B increases soluble B concentration in both the apoplasm and the symplasm. This can be attributed to the destruction of cell membrane structure through the production of reactive oxygen species (ROS) and through decrease in the phospholipid content under salinity stress ,83. ConsGenerally, B toxicity tolerance is correlated with decreased B uptake via roots and then to shoots; however, the situation can be different for tolerant genotypes. The tolerant genotypes may show B toxicity tolerance despite higher B uptake and leaf B concentrations. This feature can be attributed to the redistribution of B from the intracellular region to the apoplastic region .+/H+ antiporter, and other transporters are thought to be involved. Vacuolar Na+/H+ antiporter facilitates the movement of Na+ ions into the vacuole and may protect the cytoplasm from ion toxicity [+/H+ antiporter is overexpressed; this increases the vacuolar compartmentalization of sodium ions and thus increases the salt tolerance in plants [In plants with less damaging symptoms of combined BorSal stress, proton transporting ATPases, Natoxicity . Under sn plants ,88.+-ATPase are the proteins that produce the electrochemical H+ gradient across the membranes. This proton gradient facilitates the translocation of sodium ions and Na+/H+ antiporter out of the cytoplasm and their compartmentation into vacuoles. This in turn may increase the salt stress tolerance by decreasing sodium ion toxicity in the cytoplasm. Several studies have reported that individual salt supply enhances the H+-ATPase activity and thus the Na+/H+ antiport activity in the cell; in contrast, individual boron toxic condition inhibits the H+-ATPase activity [+-ATPase activity, although the increment can be lesser than that achieved under the individual salt toxic condition, and it may have an ameliorative effect on plants [The plasma membrane and the vacuolar Hactivity ,89,90,91n plants .Plasma membranes intrinsic proteins (PIPs) including aquaporins control the transport of water into the cells by the formation of aqueous pores in the membranes ,93,94. HStudies on aquaporin isoforms in maize showed their involvement in the transport of Na and Cl ions and in increasing the membrane permeability towards boric acid. This leads to a reduction in PIP abundance, leading to the regulation of salt and B toxicity. Consequently, PIP aquaporin levels are down-regulated in the presence of salinity with a reduction in PIP abundance. However, these reductions may vary with different isoforms. Likewise, PIP abundance towards B stress also varies in different PIP families. In some studies, transport of boric acid controlled by PIP aquaporins suggests that reduction in PIP levels might diminish the outcomes of boric acid toxicity ,22,98,992+ ion concentrations in plants seem to be a major link in the effect of salinity on B accumulation. Calcium is well known for maintaining the integrity of cell membranes [2+ uptake by disturbing K+/Na+ balance at membranes [2+ transport and vice-versa [2+ ions bind with them and form complexes that migrate faster [The total Caembranes . Salinitembranes ,102,103.embranes . Howeverce-versa ,106. As e faster . Thus, B+ content to a major extent, as it is not involved in the K+ uptake pathway [Imbalanced uptake of inorganic ions such as Na, K, Cl, and B in plants under B and salinity stress has a major role in their tolerance mechanisms . High sa pathway .It has been reported, that under high substrate B concentration, the genes encoding root-shoot aquaporins are down-regulated . This deAlthough different characteristics have been discussed related to membrane permeability and its role in providing tolerance to B and salt stress, it also largely varies from species to species and their sensitivity towards salt stress .B is mostly immobile in plants, and its movement mainly occurs through xylem depending upon the transpiration force that causes higher B concentration at leaf tips and in mature leaves ,24. ThisIn a few species, salinity has no influence on B concentrations at the base of the leaves, while it increases the B levels at leaf tips . HoweverStomatal resistance of leaves is an indicator of stress level in plants . B uptak2 within the cells that is required for photosynthesis, leading to the accretion of NADPH. Hence, oxygen becomes activated as an electron acceptor forming the superoxide radicals. Although more superoxide radicals can be produced under combined B and salt stress condition, it is followed by the formation of antioxidant molecules under BorSal treatment, leading to the reduction in toxicity symptoms  enlarges the AtNIP7;1 pore opening that positively affects the B transport activity [Arabidopsis genes, the maize tassel-less1 gene (ZmTLS1) is confirmed as a boric acid channel with its involvement in vegetative and inflorescence growth under limited B supply [OsNIP3;1 gene of rice is confined to different tissues, including vascular bundles in leaf sheaths and exo-dermal cells in roots. Its function as boric acid channel is confirmed with the reduction in plant growth under B-limiting conditions by the silencing of OsNIP3;1 RNA [The nditions . Its GFPnditions . It was nditions . AtNIP6;o phloem ,157. NIPactivity . SimilarB supply . The OsNP3;1 RNA .A. thaliana that encode BOR-type borate transporters (BOR1–BOR7) [A. thaliana under B-deficient conditions. However, its homologs in yeasts and different plant species confer tolerance towards B toxicity.Other than boric acid channels, borate exporters have a crucial role in maintaining the B concentration in cells by the efflux of B. Seven genes have been reported in R1–BOR7) ,162,163 BOR1 homologs are known to regulate B uptake by efflux from the root cells to their surroundings and, consequently, their controlled transfer to shoot. In leaves, toxicity symptoms are not always according to the leaf B content because of differential partitioning of B. However, it was suggested that, under toxic conditions, B transporters pump out B from the symplast to the apoplast, where relatively higher B concentrations can be endured. Tolerant cultivars are more efficient in pumping out this toxic B from the cytoplasm to the apoplast .Several studies have reported the role of BOR1 up-regulation in facilitating the movement of B in shoots under B deficient conditions. Recently, Aibara et al.  highlighSaccharomyces cerevisiae provide tolerance to high B concentration by exporting B out of the cell [Ta-BOR2) and barley (Hv-BOR2) showed positive associations between their expression levels and the decrease in the B concentrations in roots, specifically in the tolerant cultivars [BOR1 homologs in the cell ,165,166.ultivars ,167. AddAtBor1 in wheat, TaBOR1, differentially controls the import of B in roots and its movement into shoots [TaBOR1.1, TaBOR1.2, and TaBOR1.3, respectively, express differently under B limiting and B excess conditions. Among these alleles, TaBOR1.1 and TaBOR1.3 are upregulated under B deficient conditions; however, TaBOR1.2 is up-regulated under B toxic conditions in both roots and shoots, signifying that it might be involved in draining out surplus B from the tissues, thus providing endurance against B toxicity.The ortholog of o shoots . Even itAtBOR4 is involved in B exclusion from tissues primarily from roots, leading to excess B tolerance [TaBor2 and HvBOR2 [olerance ,169, andd HvBOR2 ,167,170.d HvBOR2 ,163. Bei+ ions) occurs in the cytoplasm of the plant cells, thereby inhibiting photosynthetic activity, protein synthesis, and other developmental processes [+ exclusion and tissue tolerance [In most of the plant species, salinity stress can be categorized into two forms, osmotic stress and ion toxicity ,171,172.rocesses . Where oolerance ,173. Pla+/K+ ratio [1 , HKT (high-affinity K+ transporters), and NHX (Na+/H+ exchangers) transporters. The over- or under-expression of these genes maintain Na ion uptake, their translocation, and homeostasis within tissues [1, NHX, and HKT transporters are responsible for the efflux of Na+ from roots, its sequestration into vacuoles, and its influx in roots along with the recovery from xylem, respectively [The disturbance of Na ions in tissues impedes the K ions in the plants. Hence, salt stress is substantially studied in the context of cytosolic NaK+ ratio . This ra tissues ,176,177.ectively ,184,185.1, which encrypts plasma membrane Na+/H+ antiporter, facilitates xylem loading in roots under limited saline conditions, thereby regulating the distribution of Na ions into shoots and leaves. However, increase in Na+ ion concentration in shoots under high saline environment inhibits their transfer from roots to shoots, thereby minimizing its loading into the root xylem tissues and thus promoting the salinity tolerance of plants [+ from the root cells to the apoplast with the help of proton gradient developed by H+-ATPase [1 under high saline conditions has been reported in Arabidopsis and other plant species [The expression of SOSf plants ,189,190.+-ATPase ,192. Thi+-ATPase ,193. The species ,196,197.1, two other proteins, SOS2 and SOS3, are found to be involved in providing salt tolerance to plants [2SOS gene encrypts a serine/threonine protein kinase and possesses an N-terminal catalytic domain and a C-terminal regulatory domain [3 contains a myristoylation location at its N-terminus and a myristoylated Ca+ binding protein, which are both necessary for developing salinity tolerance [+ binding site of SOS3 protein binds with the FISL motif of SOS2 [3–SOS2 complex phosphorylates SOS1 and, consequently, activity of the SOS1 protein increases, providing salt tolerance to plants [Other than SOSo plants . SOS2 gey domain . SOS3 coolerance . Being s of SOS2 ,202,203.o plants ,204.+/K+ homeostasis in plants. The up-regulated expression of these genes under high saline conditions occurs with the increase in Na+ concentration in the vacuoles of leaves that causes the discharge of Na from xylem vessels, thus mitigating the salinity effects in leaves [+-K+ co-transporters, respectively [Arabidopsis plant, Class I transporters HKT1 mutants were found to be susceptible to salinity stress with higher sodium concentration in leaves [AtHKT1 intervenes with Na+ influx into roots [+ from xylem vessels [OsHKT2 that belongs to Class II transporters was reported to be involved in both Na+ and K+ influx into root cells of plants under salinity stress to provide salt tolerance [TmHKT1 of Triticum monococcum extracts Na+ from the xylem and regulates movement into leaves [HKT transporters promote unloading of Na ions from xylem into parenchymal root cells and loading of K ions from roots into xylem ,208,209.n leaves ,210,211.ectively ,213. In n leaves . Some stto roots , while o vessels . In riceolerance ,216. TmH+/H+ exchangers) transporters play a crucial role in this process [+/K+ homeostasis [AtNHX1 and AtNHX2 of Arabidopsis are determined to be involved in K+ influx into vacuoles, maintenance of vacuolar pH, and compartmentalization of Na+ into vacuoles [Arabidopsis, wheat, maize, rice, Brassica, etc. [To alleviate Na ion concentration in the cytoplasm, plants sequester Na+ in vacuoles, and NHX (Na process . The inc process ,218,219.eostasis . AtNHX1 vacuoles . The oveca, etc. ,223,224.Different transporters have been studied under individual B and salt stress conditions. Some of these transporters are common under both stress conditions and regulate the toxicity levels by up- or down-regulation of their expression. However, there is a lack of information on their differential expression under a combined stress condition.ZmPIP2 and ZmPIP1 levels in comparison with the individual boric acid and NaCl stress, respectively [HvPIP1 and HvPIP2, are recognized as B and salt transporters [HvPIP1 and HvPIP2 activity was found under salt and combined stress in roots as compared to that in B stress alone. However, these isoforms are up-regulated in leaves, thereby maintaining the cellular water level and controlling the water uptake from the soil.Although there have been distinct presumptions on the effect of BorSal stress on plant growth and development, both stress conditions are believed to affect water movement in plants. Thus, aquaporins can be a connecting link in the tolerance mechanism. Aquaporins facilitate the exchange of B through the plasma membrane under high B supply  that is ectively . In barlsporters . A signi1 gene in roots and leaves under saline conditions has been reported in different plant species such as alfalfa [Medicago [1 is responsible for removing Na from the roots under saline stress. However, when the salt stress is accompanied by B stress, either other genes mediate the tolerance mechanism or B accumulation in shoots under high B concentration might reduce the movement of B from root to shoot.The up-regulation of the SOS alfalfa , MedicagMedicago , wheat [Medicago , and maiMedicago . However1, NHX transporters that move Na+ into vacuoles are not much affected under BorSal stress conditions [Similar to SOSnditions . Howevernditions . This conditions .BOR transporters with the function of expelling B out of root cells and symplast are remarkably down-regulated in maize under combined stress as compared to that under individual salt and B stress. The lesser B content in the shoot of tolerant cultivars with the down-regulation of BOR genes shows that their expression level controls the B uptake and, consequently, its movement into shoots ,230.From different studies, it can be understood that a combined B and salinity stress condition does not always have a positive effect on plant development as compared to individual stresses. Hence, the involved genes should be studied at the molecular and the cellular levels by comparing the mechanism under two conditions—one where combined stress has a positive effect on plants and another where combined stress has a negative effect on plants. Moreover, most of the gene expression-based BorSal stress studies have been performed on tolerant cultivars. The comparison of gene regulation in tolerant and susceptible cultivars may highlight new pathways in B and salt transport in plants. Additionally, the same plant species demonstrated different outcomes under BorSal stress in different experiments. Although the growth conditions involving soil, temperature, B, and salt dosages were different in these experiments, the genetic variation of cultivars cannot be neglected. Hence, it is suggested to compare the gene expression results of different cultivars developed under the same high B and salt stressed growth environment. The genetic variation of these cultivars may give new insights into the responsible pathways in terms of expression of the involved genes. The differential regulation of a gene can be responsible for the susceptibility of a cultivar to a particular stress condition. A hypothesis can be considered that, if a gene is up-regulated or vice-versa under BorSal stress, stimuli that can oppositely express that gene should be investigated. For example, if any gene A is down-regulated in a susceptible cultivar under BorSal stress, the factors that can up-regulate its expression in the susceptible cultivar should be discovered. Furthermore, it should also be investigated whether the upregulation of that gene A in susceptible cultivar improves its tolerance to BorSal stress. The purpose of this review was to emphasize that BorSal stress should be considered as a novel stress condition, and the information provided in this review can be used to understand the gaps in this research area. These gaps should be closed so that crops can be grown under combined B and salt-stressed agricultural fields and yields can be elevated.


The increasing use of personal listening devices (PLDs) has been accompanied by a rise in the prevalence of hearing loss (HL) in younger age groups. However, there have been few reports on the relationship between HL and leisure noise exposure (LNE) in adolescents. The purpose of our study was to investigate the prevalence of HL in students attending general middle and high schools and to identify factors affecting HL prevalence. A total of 2,879 nationally representative adolescents in the first years of middle and high school underwent audiometric testing and otological examinations, and completed questionnaires, from June to December 2016. A speech-frequency hearing loss (SFHL) was considered present when the pure tone averages (PTAs) at 0.5, 1, and 2 kHz were ≥ 15 dB and a high-frequency hearing loss (HFHL) was considered present when the PTAs at 3, 4, 6, and 8 kHz were ≥ 15 dB. About 17% of Korean adolescents exhibited at least slight HL. The prevalence rates of SFHL and HFHL in the poorer ear were 11.6% and 10.3%, respectively, among Korean adolescents. The use of local area network (LAN) gaming centers and an experience of being asked by others to lower earphone volume were associated with both SFHL and HFHL. It is important to avoid excessive LNE to prevent adolescent HL. Additionally, SFHL or HFHL in the poorer ear was associated with lower academic performance. Noise-induced hearing loss is a hearing impairment caused by exposure to loud sounds, and initially affects the high-frequency hearing threshold. Previously, noise-induced hearing loss that was attributable to occupational noise exposure was of major concern, and hearing loss (HL) caused by leisure noise exposure (LNE) was considered to be relatively unimportant , 2. AlthA study from the USA reported that the prevalence of HL among US adolescents increased from 14.9% in 1988–1994 to 19.5% in 2005–2006. Although no significant relationship between noise exposure and HL was found, an influence of noise may be inferred by the reported increase in high-frequency hearing loss (HFHL) . AnotherThus, it has not yet been determined whether noise exposure affects adolescent HL. LNE is difficult to quantify and HL attributable to LNE varies from a temporary threshold shift to permanent HL. However, if noise exposure affects adolescent HL, early detection of HL and avoidance of additional noise exposure may be the most important factors to prevent further exacerbation.In this study, we investigated the prevalence of HL and the associations between HL and noise-related factors, including specific types of LNE that adolescents are typically exposed to, as well as self-rated academic achievement in adolescents in South Korea.We performed a cross-sectional, complex sampling survey of first-year middle and high school students, except those attending special schools for disabled children. The sampling frame was the 2015 yearbook of educational statistics prepared by the Korean Educational Statistics Services .We sought to enroll a total of 3,100 students from 124 middle schools and 124 high schools, based on the prevalence of HL in Korean adolescents, the estimated association between noise and HL, and an expected 80% response rate , 16. TheThe survey was performed from June to November 2016. After distributing written information to students and parents, we evaluated only those who provided written informed consent of themselves and their parents. This study was approved by the Institutional Review Board of Seoul National University Hospital (No. 1604-086-755).Students and parents were asked to complete separate questionnaires. Both questionnaires were based on Forms 1–2 and 1–3 of the Enforcement Rule for Health Screening at School of the School Health Act of South Korea, and those of the Korea Nutrition and Health Examination Survey .The student questionnaire explored gender, age, any history of a disorder of the ear, nose, or throat, subjective hearing status, and any tinnitus. Participants answered “yes”, “no”, or “I don’t know” to the following question about tinnitus: “Have you ever experienced tinnitus, such as beeping, ringing, or buzzing, in the past year?” Questions pertaining to personal listening devices (PLDs) concerned usage of a PLD , usage period (“How old were you when you started using a PLD?”), requests to turn down the volume (“Have you ever been asked to turn down the volume on your PLD?”), and the subjective volume intensity , based on a previous study by Cone et al. . In termPure tone audiometry was conducted using an AD229b diagnostic audiometer  in soundproof booths inside a mobile vehicle by four experienced audiologists. Prior to audiometry, otological examinations were performed by trained, experienced otolaryngology residents using otoscopes. Factors affecting hearing, such as external ear anomalies, ear wax, retraction of the tympanic membrane, cholesteatoma, and middle ear effusion were recorded; modifiable factors  were managed prior to the hearing tests.The tested frequencies ranged from 0.5 to 8 kHz, i.e., 0.5, 1, 2, 3, 4, 6, and 8 kHz. Testing began at 1 kHz, followed by 2, 4, 8, 1, 0.5, and 0.25 kHz. The participants started the test by listening to a 30 dB tone; the test tone was reduced by 10 dB or increased by 5 dB according to the presence or absence of a response. The hearing threshold was defined as the lowest intensity level (in dB) at which the participant responded to 50% of the stimuli. When the hearing threshold was ≥ 25 dB at any frequency (0.5–8 kHz), the bone conduction threshold to distinguish between sensorineural and conductive HL was determined. The severity of HL was based on the poorer threshold for unilateral HL and the better threshold for bilateral HL, as described previously . We defip-value < 0.05.First, we calculated the unweighted frequencies and weighted prevalence rates of SFHL and HFHL; this was deemed appropriate given the multi-stage, complex sampling design. Second, we used a multivariate logistic regression model to calculate odds ratios (ORs) for the associations between HL and various factors. The model included demographic characteristics ; known risk factors ; tinnitus status ; and associations between HL and noise sources . To estimate the association between leisure time noise exposure and HL with consideration of known risk factors, noise sources were not included as model covariates. We adjusted for potential confounders (known risk factors for HL) including age, gender, household income, smoking status, alcohol consumption, any history of ear infection or rhinosinusitis, and any family history of HL . Third, Overall, 109 of the 248 schools contacted agreed to participate in the survey, with 3,013 students agreeing to complete the questionnaires. Of the 3,013 participants, 134 were excluded because of incomplete data. A total of 2,879 students underwent audiological and otological examinations (95.6%). The demographic characteristics of these 2,879 participants are summarized in The prevalence rates of SFHL and HFHL were 11.6% and 10.3%, respectively, in Korean adolescents. In total, 17.2% exhibited either SFHL or HFHL. The prevalence of any HL  were 17.9% and 16.5% among middle and high school students, respectively. The prevalence rates of any HL at high frequencies were 10.5% among middle school students and 10.2% among high school students. The prevalence rates of any HL at speech frequencies were 12.7% and 10.4% among middle and high school students, respectively. The prevalence of slight HL was 16.0% and that of serious HL was 1.2%. The detailed data are shown in We constructed multivariate logistic regression models to analyze the associations between HL and related factors . All anap = 0.003). However, no significant association was found between LAN use time and SFHL. The prevalence of HFHL was affected by the average use frequency  and time spent per visit .Analysis of LAN gaming center use and HFHL prevalence showed a positive correlation between cumulative hours spent in gaming centers and HFHL prevalence . CompareHL affected school performance. In children with ≥ 15 dB of HFHL or SFHL, the odds of lower academic performance were 1.33-fold  and 1.47-fold , respectively, higher than those of other children .We found that the prevalence of HL was 17.2% overall, and 17.9% and 16.5% in middle and high schools, respectively. The extent of HFHL was 10.3%. LNE in noisy LAN gaming centers and setting earphone volume to levels causing others to ask that the volume be reduced affected HFHL. Both SFHL and HFHL were inversely associated with self-rated academic performance.Currently, many adolescents use smartphones, which are the most common PLDs. In 2011, adolescent Korean smartphone users constituted 12.8% and 21.7% of all middle and high school students, respectively, but those figures rose to 86.6% and 90.2% in 2015 , 20. The“Do you use portable music and video playback devices, such as smart phones, MP3 players, or PMPs?”Participants who had a smartphone for a purpose other than listening to music were also likely to answer “yes” and be misclassified as PLD users, which obscured the relationship between PLD use and HL. However, the following questions that concerned the precise pattern of PLD use showed that the requests to lower the volume seemed to be associated with greater HL. Therefore, the volume of LNE may be closely related to HL. This volume ranges from 50–100 dB. Use of PLDs at volumes > 80 dB LAeq increased hearing thresholds and affected syllable identification in a noisy environment [We investigated the relationships between HL and several types of LNE: LAN gaming centers, karaoke rooms, and concert auditoria. The prevalence of HFHL was higher in adolescents using LAN gaming centers, in contrast to the findings of a recent report indicating that no significant relationship was evident between HL and the maximum equivalent LNE, such as time spent in nightclubs or music venues, at concerts or festivals, and listening through headphones . Shin etIn contrast, subjects who attended concerts had a lower prevalence of HFHL than those who did not. Of 257 subjects who attended concerts in the past year, 154 (65.3%) went to a single concert, in marked contrast to the number of visits to LAN gaming centers. Occasional exposure to concert noise is less likely to affect hearing than repetitive noise. Also, the concert noise level would vary by the featured music genre. Last, those who attended concerts had higher family incomes than others; the negative association between concert attendance and HFHL is explained by the difference in income level.Both HFHL and SFHL were negatively related to household income, consistent with the findings of previous studies. The National Health and Nutrition Examination Survey of 2005–2006 reported that school-aged children living in poverty had a higher prevalence of HL than other groups . A KoreaWe found that even slight HL among school-aged children is significantly associated with self-rated academic performance after adjusting for gender, grade, and household income. Previous studies found that educational performance was (negatively) associated with mild bilateral or unilateral HL –28. BessHearing screening tests have been conducted in preschool and/or school-aged children in many countries , 36. PurOur study had certain limitations. Some questions required long-term memory recall  and students often do not know their exact medical history. Thus, more complicated questions were answered by the parents, while students were asked about their major medical history in the past year. In addition, causal inferences cannot be drawn due to the cross-sectional nature of the study design. The school participation rate was low because of the complexity of the methods, which required active involvement of students, parents, and teachers. The audiological tests were conducted at the mobile soundproof booth in schools for convenience; this may have caused an increased prevalence of slight HL at the speech frequencies, due to uncontrolled environmental vibrations and excessive noises from the nearby playground or school buildings.Despite these drawbacks, this study is noteworthy in that it assessed, through detailed audiometry, the actual prevalence of HL and HFHL in adolescents who were regarded as normal by conventional hearing screening testing. In addition, the authors analyzed the association between various types of LNE and HL. As LNE in adolescents is increasing, and is expected to increase further in the years to come, the results of our study constitute useful information for preventing HL in adolescents.About 17% (one-sixth) of Korean adolescents exhibit at least slight HL. Frequent exposure to loud, high-intensity leisure noise can affect the hearing threshold. HL may be associated with poor school performance. It is important to avoid excessive exposure to leisure noise when seeking to prevent HL in adolescents.S1 Text(PDF)Click here for additional data file.S2 Text(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.