Globally, 5.82 million deaths occurred among children under the age of five years in 2015 and injury specific mortality rate was 73 per 100,000 population. In India, injury specific mortality rate is around 2.1 per 1000 live births contributing to 4% of the total under 5 mortality rate. This study aims to estimate the burden and understand factors associated with unintentional injuries among children aged 1–5 years residing in urban slums of Vellore, southern India. We also attempted to assess the hazards posed by the living environment of these children and study their association with unintentional injury patterns.This cross-sectional study was conducted in eight urban slums of Vellore, southern India and primary caregivers of children aged 1–5 years were interviewed with a questionnaire to obtain the details of injuries sustained in the past three months. Environmental hazard risk assessment was conducted at places frequented by these children and their scores calculated. Baseline prevalence and incidence rates of unintentional injuries were estimated. Multivariate logistic regression and poisson regression analysis were performed to examine factors associated with unintentional injuries and repeated injuries respectively. Association between environmental hazard risk and unintentional injuries was estimated.N = 662). Bivariate analysis revealed that children of working mothers  and children from overcrowded families  had increased odds of sustaining unintentional injuries. Multivariate regression analysis revealed that children from overcrowded families had increased odds of sustaining unintentional injuries . Boys  and children from overcrowded families  were at increased risk of having repeated injuries. There is an increase in incidence rate of injuries with an increased environmental hazard risk, although not statistically significant.Prevalence of unintentional injuries was 39.1% (95% CI 35.4–42.9%) and incidence rate was 16.5 (95% CI 14.7–18.3) per 100 child months  contains supplementary material, which is available to authorized users. Globally, 5.82 million deaths occurred among children under the age of five years in 2015.  in India in 2013 is 52.7 per 1000 live births and injury specific mortality rate is around 2.1 per 1000 live births contributing to 4% of the total U5MR WHO . StudiesIn India, unplanned urbanization and rise in informal settlements are leading to an increase in urban slums thus leading to high population density and overcrowding , population density of 42,000 per kmAn indigenously developed Environmental hazard form (EHF) was used to assess the risk of different types of injuries such as falls, burns , road traffic injuries, poisoning, drowning, and mechanical hazards from household objects. For risk assessment, a base score (ranging from 0 to 4) was assigned based on the perceived severity for each type of injury mentioned above. A modifying factor score for each type of injury (0–4) was assigned based on the child’s accessibility to the injury risk. The end hazard score for each injury type was calculated by multiplying the base score and the modifying factor score  the score was 4 × 2 = 8″) . The overall prevalence and incidence rates of UI per 100 child months with 95% confidence intervals were estimated based on injuries over a three month recall period which is three months prior to the date of interview. Bivariate analyses were performed to examine associations between socio-demographic factors and unintentional injuries among children. All socio-demographic factors with A total of 662 children were surveyed of which, 321(48.5%) were boys and 341(51.5%) were girls. Majority, 611(92.3%) belonged to lower socioeconomic group and 356 (53.8%) were Hindus. The median (IQR) family size was 5(4–7) and 361(55%) families had ≤2 children in the house. Predominantly, 499 (75.4%) families had ≤2 rooms in the house and 273 (41.3%) families lived in pucca houses. The mean (SD) years of schooling among mothers was 7.6 (2.8) and 87 (13%) mothers of study children had no formal schooling. The mean (SD) age of mothers was 26.4 (4.2) years and about 22% (146/662) of them were gainfully employed   over a three-month recall. The prevalence of UI was 42.6% (137/321) among boys and 35.8% (122/341) among girls and this difference was not statistically significant. Proportion of children sustaining UI in the age groups 13–24; 25–36; 37–48 and 49–60 months were 33.7% (95% CI 27.3–40.7); 40.3% (95% CI 33.0–47.5); 40.9% (95% CI 33.5–48.6) and 43.1% (95% CI 35.0–52.0) respectively. Even though the proportion of children getting injured were increasing with age, this association was not statistically significant . However, there were no statistically significant gender differences in burns and road traffic injuries . Children in the age group of 4–5 years had a high risk of injury  with an incidence rate of 17.4 (95% CI 13.6–21.9) per 100 child months  of sustaining unintentional injuries than children whose mothers stayed at home. Similarly, children from overcrowded families had significantly higher odds of having an unintentional injury . Other socio-demographic factors including presence of siblings, maternal age and education level; paternal age, education, occupation; family size and income were not significantly associated with higher risk of unintentional injuries Table . MultivaAny child sustaining more than one unintentional injury during the three-month recall period was considered to have repeated UI. Poisson regression analysis for assessing the risk of having repeated UI has revealed that, children from overcrowded families experienced higher risk of repeated unintentional injuries  after adjusting for age, gender, presence of siblings, maternal occupation, housing type and overcrowding. Boys had significantly increased risk of having repeated injury compared to girls . Older children (age > 2 years), the presence of siblings, working mothers, and poor housing conditions had higher risk of repeated UI but not at statistically significant level (Table The mean (SD) of total environmental hazard score (EHS) was 24.314.5). The highest hazard score was present at babysitter/caregivers home 28.6(12.3) followed by homes of children 28.1(16.8). Play areas had a hazard score of 23.1(13.2) and least hazardous were schools with a score of 18.8(9.7). Based on the 33rd and 66th percentiles of the total EHS, children exposed to areas with scores less than or equal to 18.3 were considered to be at low risk, between 18.4 and 28.5 to be at moderate risk and more than 28.5 to be at high environmental risk for sustaining injuries. Among all the study children, 209(32%) were exposed to low-environmental risk, 218(33.5%) to moderate- and 225(34.5%) children to high environmental risk. The proportions of children sustaining UI were 35.8%, 39.5% and 42.3% in the low, moderate and high environmental risk categories and this rising trend was not statistically significant , even when the perception of hazard risk is high and maternal supervision is high, the rate of UI remains high , the environment they live in and various socio-demographic factors; all these factors are interrelated and influence each other  could possibly be used as a tool to assess the risk of UI at homes and other places frequently visited by children, after it has been validated with a larger sample size. Recent initiative by the Government of India for building smart cities with planned urbanization should be addressing the environmental hazards posed to the communities. However, policy decisions for community-based interventions with combined engineering, environmental measures need to be implemented to reduce the environmental hazards to children while further research is recommended to ascertain the behavioral and psychological components.Additional file 1:Questionnaire for childhood injuries (DOCX 21 kb)Additional file 2:Table S1. Example of calculating environmental hazard score for a subject at one location. Table S2. Distribution of different types of injuries as per gender (DOCX 14 kb)Additional file 3:Environmental Hazard Observation Form (DOCX 22 kb)


With an estimated 174 million undiagnosed cases of diabetes mellitus worldwide and 80% of them occurring in low- and middle-income countries an effective point-of-care diagnostic tool is key to fighting this global epidemic. Glycated hemoglobin has become a reliable biomarker for the diagnosis and prognosis of diabetes.We assessed two point-of-care (POC) analyzers in multi-ethnic communities of the Amazon Rainforest in Peru where laboratory-based glycated hemoglobin (HbA1c) testing is not available.203 venous blood samples were tested for HbA1c by Afinion and DCA Vantage analyzers as well as a Premier Hb9210 high-performance liquid chromatography (HPLC) method as the reference standard. The coefficient of variation (CV) of each device was calculated to assess assay imprecision. Bland-Altman plots were used to assess bias. Ambient temperature, humidity, and barometric pressure were also evaluated for their effect on HbA1c results using multivariate regression.p < 0.001), as did the DCA Vantage (mean difference = +0.32% [4 mmol/mol] p < 0.001). Temperature and humidity were not related to HbA1c; however, barometric pressure was associated with HPLC HbA1c results for the Afinion.There was a wide range of HbA1c for participants based on the HPLC test: 4.4–9.0% (25–75 mmol/mol). The CV for the Afinion was 1.75%, and 4.01% for Vantage. The Afinion generated higher HbA1c results than the HPLC (mean difference = +0.56% [+6 mmol/mol]; Imprecision and bias were not low enough to recommend either POC analyzer for HbA1c determinations in this setting. Diabetes mellitus is a global health crisis with an estimated 174 million people undiagnosed . In addiThe use of HPLC to measure HbA1c in rural LMICs settings is largely untenable due to economic and geographic barriers. Cost of sample collection, transportation, and processing is high, locations with appropriate laboratory standards are not easily accessible, and protocols for storage and transport of samples to central labs face significant challenges . Point-oThe Afinion™ AS100 Analyzer (Alere Technologies) and DCA Vantage™ Analyzer  are National Glycohemoglobin Standardization Program (NGSP) certified POC devices for evaluating HbA1c . Both deA cross-sectional study was performed in communities surrounding the Amarakaeri Communal Reserve in the southern Peruvian Amazon region of Madre de Dios (MDD). MDD is home to over 137,000 people, with several indigenous tribes disbursed throughout the area . MedicalThis study was embedded within the larger Amarakaeri Reserve cohort study, designed to evaluate the impact of natural resource extraction  on human and environmental health. The cohort study enrolled 1,122 households from 23 communities surrounding the reserve that are affected directly and indirectly by mining activities  is a POC device that utilizes boronate affinity chromatography to measure the total percentage of glycation using a 1.5 μL sample of blood . It has 12The DCA Vantage™ Analyzer  uses an immunoassay based on antibodies binding to glycated hemoglobin tetrapeptide or hexapeptide molecules using a 1.0 μL sample of blood . It has Households with at least one woman of child-bearing age (15–49 years) were eligible for selection in this study. Community maps were obtained for Huepetuhe, Quebrada Nueva, and Quince Mil to draw a random sample. Since there were less than 75 households in Caychihue, Puquiri, and Setapo, each household with a woman of child-bearing age was approached to participate. Selected households that met the eligibility criterion were introduced to the study by trained fieldworkers, and family members were asked if they would like to participate. If consent was obtained, surveys were administered and biomarker samples were collected from the mother of the sentinel family unit, her spouse, and any children aged 12 and under. Venous blood samples were collected in K2 collection tubes containing edetic acid (EDTA) as a preservative. The tubes were labeled with the patient’s name, unique identifying number, and date, then placed in a Credo Cube™ cold box to store the samples between 2–8°C for up to ten days. Capillary blood samples were also drawn and anemia testing was performed with a HemoCue® POC device.Batches of venous blood samples were delivered approximately twice per week to the town of Mazuco, where they were tested with the Afinion and DCA Vantage POC analyzers. Per manufacturer recommendations, before commencing tests on the participant samples each POC analyzer was initially verified to be in proper working condition by testing controls purchased from the manufacturers. Since HbA1c measurement can be affected by high temperature, the heat-sensitive color pads on the front of each box of DCA Vantage reagents was checked to ensure that the maximum temperature limit was not exceeded during transportation and storage. In addition to recording data on the HbA1c level of each sample, data was collected on the temperature, relative humidity, and barometric pressure of the room at the time of testing using the Ambient Weather WS-110 Wireless Weather Station. Furthermore, a single blood sample near the 6.5% (48 mmol/mol) diagnostic threshold for diabetes was measured 14 consecutive times using both POC devices.After obtaining HbA1c levels from the POC analyzers, the samples were stored at a 20°C cold chain and shipped to the Medlab Laboratory in Lima, Peru, where HbA1c levels were obtained using HPLC via the Premier Hb9210™ Analyzer . Control samples with HbA1c values of 5.5% and 11.5% were tested daily on the Premier Hb9210 to ensure proper calibration of the analyzer. All control testing measurements were within a 0.2% and 0.3% deviance for the 5.5% and 11.5% controls, respectively.The analytical objectives were to measure imprecision of each POC device and to compare their HbA1c results to those obtained from the Premier Hb9210 analyzer. HbA1c was reported in Diabetes Control and Complications Trial (DCCT) aligned units (%) and converted to International Federation of Clinical Chemistry (IFCC) units (mmol HbA1c per mol unglycated hemoglobin) using the following master equation :Imprecision was evaluated from the sample of 14 repeated measurements using the coefficient of variation (CV), calculated as:s is the sample standard deviation and where recision .POC and HPLC results were compared by examining Pearson correlation coefficients from univariate regressions of paired samples and paired t-tests. Bland-Altman plots were used for bias assessments. Univariate regression was performed to test whether the bias was constant across the range of HbA1c concentrations.Ambient temperature, humidity, and barometric pressure were evaluated for their effect on HbA1c results. A multivariate regression predicting HPLC’s HbA1c with results from each POC device and the ambient measurements was performed. Backwards selection procedures were used to remove non-predictive variables from the model. All statistical analyses were performed with STATA v13.1 (StataCorp LP).Approval for this study was obtained from the Institutional Review Board of the Universidad Peruana Cayetano Heredia. Children 12 and older provided assent, and all adult participants and parents of child participants provided informed consent and were advised on the research aims and objectives, rationale, expected benefits, and rights of participants.HbA1c results using the Afinion and DCA Vantage POC analyzers and the Premier Hb9210 HPLC analyzer were obtained for 203 individuals. 137 participants were female (72%) with an average age of 36.6 years (range 12–75 years). Hemoglobin concentrations were obtained for 196 of the 203 participants, with 38 individuals (19.3%) having levels under 120 g/L for women and 130 g/L for men, indicating anemia. Anemia was more common in women (21.4%) than men (13.7%). Data about malaria was available for 198 participants, and 11 individuals (5.6%) reported being diagnosed with malaria in the past year.The HbA1c values ranged from 4.4% (25 mmol/mol) to 9.0% (75 mmol/mol) per the HPLC analyzer  for the Afinion, and 6.37% (46 mmol/mol) for the DCA Vantage. The sample standard deviation was 0.117% for the Afinion, and 0.255% for the DCA Vantage. The corresponding CV was lower for the Afinion (1.75%) than the DCA Vantage (4.01%).The correlation of HbA1c values between the HPLC and POC analyzers were slightly higher for the DCA Vantage than Afinion. Correlation with the Afinion was 0.92, while correlation for the DCA Vantage was 0.93 Figure , b.p-value < 0.001 by paired t test. The bias between the DCA Vantage and the HPLC analyzer was +0.32% (+4 mmol/mol) (95% CI 0.30% to 0.35% [3–4 mmol/mol]), p-value < 0.001. The bias observed was constant across the range of HbA1c concentrations for the DCA Vantage (p = 0.190), but not for the Afinion (p < 0.001), where bias increased as HbA1c levels decreased.Bias relative to the HPLC test for the Afinion was +0.56% (+6 mmol/mol) (95% CI 0.53% to 0.59% [6 mmol/mol]), The Bland-Altman limits of agreement (LOA) between the Afinion and the HPLC analyzer were 0.16% to 0.97% (2 to 11 mmol/mol), indicating that an individual from the studied population would have an expected difference within this range Figure . The LOAp = 0.022).Temperature ranged from 23.9–28.4°C (median 26.4°C), humidity ranged from 0.70–0.91% (median 0.83%), and barometric pressure ranged from 28.33–28.48 inHg (median 28.48 inHg). From the regression analysis, temperature and humidity were not related to HbA1c results for either POC device; however, barometric pressure was associated with the Afinion HbA1c. A one-unit increase in barometric pressure was associated with a 0.342% (DCCT units) increase in HbA1c  . We founA review of the literature reveals that the CV we found for the Afinion is consistent with values reported in other studies, which range from 0.5%  to 3.1% 56202122616202324255617182325276162326For communities in the Peruvian Amazon, access to laboratory diagnostics for clinical care remains lacking. POC testing represents an opportunity for health posts to obtain clinical information that would otherwise be unavailable for patients due to financial and transportation constraints. Information to help diagnose and manage treatment for patients with diabetes will be particularly important in South and Central America, where it is projected that the number of people with the disease will increase 65% by 2040 [While we were able to demonstrate the bias and imprecision of two POC analyzers compared to an NGSP-certified HPLC method, we acknowledge there were a few limitations with our study. First, interference from hemoglobin variants was not evaluated. The Afinion and DCA Vantage are unaffected by most common variants  but HbF levels greater than 10–15% can interfere with results . Also, sIn short, in settings such as hard-to-reach populations and indigenous groups where there is a high need to initiate diabetes prevention efforts, POC devices provide the appeal of simplicity of use and can potentially lower diabetes-related costs compared with laboratory-based methods. However, in this study we demonstrated that the DCA Vantage did not meet generally accepted performance criteria for HbA1c measurement precision, and both analyzers showed a large positive bias that may lead to unnecessary medicalization if utilized in this setting.


Tumor lysis syndrome (TLS) is common complication of acute lymphoblastic leukemia (ALL). It is characterized by presence of two or more of hyperkalemia, hyperuricemia, hyperphosphatemia and hypocalcemia. TLS may cause acute kidney injury (AKI), arrhythmias and seizures. Our objective was to determine the frequency of TLS and its biochemical abnormalities in children with ALL.A retrospective study on 91 children, aged 2-13 years with ALL was carried out in Nephrology and Oncology departments of National Institute of Child Health, Karachi from January 2016 to December 2017. Patients already received chemotherapy were excluded. Data including risk categories, immunophenotyping, laboratory parameters like complete blood picture, serum creatinine (SCr), potassium(K), calcium (Ca), phosphorus(P) and uric acid (UA) on day 0,3 and 7 after chemotherapy were collected. Data analyzed on SPSS using descriptive statistics. Independent t- test was applied to compare means and P- value<0.05 was taken as significant.9) where as hyperleukocytosis and blast cells were observed in 20.87% and 73.6% respectively. Comparing the biochemical parameters of ALL, the difference in SCr from D0 vs D3  was significant (p=0.001). Similarly, difference in UA , SP and for K (p=0.038) was significant. There was no difference in Ca from D0 vs D3 (0.092) and D7 (0.277). TLS was found in 62.6% children, it was chemotherapy induced in 72% and spontaneous in 28%. Clinical-TLS was observed in 14% and all CTLS had AKI. Hyperuricemia and hyperphosphatemia were the most common biochemical abnormalities in laboratory-TLS and CTLS.Ninety-one children with mean age of 6.39±3.08 years were studied. Male were 57% and 43% female. High risk ALL were 61.5%. Pre –BALL were 82.4% and 17.5% had T-cell ALL. All patients had anemia (hemoglobin7.69±2.66 g/dl) and thrombocytopenia (43.61± 18.6 x10TLS was found in 62.6% despite preventive measures. Early recognition and treatment is essential to avoid morbidity and mortality. Tumor lysis syndrome (TLS) is an oncological emergency resulting from massive lysis of malignant cells and clinically characterized by renal failure, seizures and cardiac arrhythmias that requires early recognition and management.The massive lysis of lymphoblastic cells releases intracellular metabolites like potassium (K), uric acid (UA), phosphate (P) and products of protein and purine metabolites into systemic circulation. These high effluxes of metabolites result in abnormal accumulation in the blood and exceed the capacity of renal clearance and results hyperkalemia, hyperuricemia, hyperphosphatemia and secondary hypocalcemia. Hyperuricemia is the initial and most common abnormality, induces acute renal injury by intra-renal uric acid crystallization and It aggravates precipitation of calcium phosphate crystals in the renal tubules. This crystal nephropathy causes inflammation, obstruction and renal tubular damage ultimately manifesting as acute kidney injury. Hyperkalemia may also be aggravated by crystal nephropathy induced AKI and may cause serious dysrhythmias and death. Hypocalcemia may lead to muscle cramps, tetany, seizures and dysrhythmias. There is concomitant rise of lactic dehydrogenase because of rapid cell turn over in ALL.4AKI may occur in ALL in absence of TLS due to renal infiltration by blast cells, infections and volume depletion.More aggressive nature, rapidly proliferating tumor, with high leukemic burden and highly sensitive to drug treatment and high-risk patients are more likely to develop TLS.7Acute lymphoblastic leukemia is the most common hematological malignancy and accounts for 75-80% of all childhood leukemia.9ALL is most aggressive malignancy associated with high mortality 11.5%-24%) in Pakistan..5%-24% i1Since TLS is an acute emergency so early identification of high-risk patients, appropriate prophylactic interventions like hyper hydration, use of allopurinol, rasburicase and strict monitoring for its clinical complications like arrhythmia, AKI and central nervous system (CNS) manifestations in leukemic patients undergoing chemotherapy may help to decrease frequency, severity of TLS and improve outcome.5There are number of local studies on various aspects of ALL but very few on incidences of TLS in children.11Our pediatric oncology unit is functioning for last 15 years with an average of 500 patients are registered per year and more than 50% among them are suffering from ALL. We want to share our retrospective data of last two years, focusing on this serious complication of ALL in children.The objective of this study was to determine the frequency of TLS in children with ALL and various biochemical abnormalities in children with TLS.This retrospective cross-sectional study was conducted on 91 children with ALL who were managed in the Department of Pediatric Oncology, National Institute of Child Health, Karachi, from January 2016 to December 2017.Laboratory TLS was defined when two or more of the following biochemical abnormalities were present (i) hyperuricemia (ii) hyperkalemia (iii) hyperphosphatemia (iv) hypocalcemia, Clinical TLS was diagnosed if child had abnormality in any one end organ  along with lab TLS.Acute kidney injury was defined if more than 50% rise in serum creatinine (SCr) from baseline or SCr was more than 1.5 time upper limit of normal.Patients were categorized according to The National Cancer Institute (NCI) criteria into standard risk and high -risk groups, based on age at diagnosis, initial white blood count (WBC), central nervous system or testicular involvement .12Patients were classified into precursor -B leukemia (pre-B Cell) and precursor T-cell leukemia (pre-T-Cell), based on flow cytometry and immunohistochemistry.2/hour)and allopurinol as inhibitor of UA synthesis (10 mg/kg/day in 3 divided doses) in cases with high UA levels was started at least 24 hours before and 4 days after chemotherapy.Tumor lysis prophylaxis with hyper hydration  was calculated using Schwartz formula from patient’s height and SCr.13Blasts cells(>5%) in peripheral smear, lactic dehydrogenase (LDH) level, type of ALL based on immunophenotyping and risk stratification group were also recorded. Ethical approval was taken from hospital ethical committee.Data collected included bio-data, anthropometry, various clinical and laboratory parameters from hospital record in the predesigned proforma. The laboratory parameters included complete blood picture, UA, P, Ca, SCr and K measured as baseline (day 0) then daily after initiation of chemotherapy till 7For numerical variables like age, S.Cr, S.K, mean±SD was used and categorical variables like gender, frequency was used. Data was entered in SPSS version 20. Independent t- test was used for comparison of means. P value <0.05 was considered as significant with confidence interval of 95%.2.Majority (82.41%) had Pre-B ALL and 16 (17.58%) had T-cell leukemia. On risk stratification, 56 (61.53%) patients were in high risk group whereas 35 (38.46%) in standard risk leukemia.Data of 91 patients were included in this study. Baseline characteristics of children with ALL are shown in 9/L) in 15(16.48%) and standard (wbc < 50x109/L) in 56(61.53%). Similarly, all patients had thrombocytopenia with a mean platelet count 43.61± 18.6 x109and majority (61%) had thrombocytopenia (<50x109/L). Sixty-seven(74%) were diagnosed as ALL on the basis of presence of blasts cell in the peripheral smear and rest on bone marrow.Hematological findings  revealed2respectively.Table-IIBase line biochemical parameters and renal functions in children with ALL are shown in thday showed that mean±SD levels of UA, P and Ca was3.56±1.42, 4.13±1.07and 9.16±0.84mg/dl respectively whereas serum K was 3.76±0.51meq/L. The mean serum Cr was 0.44±0.22 mg/dl. There was significant difference (p=0.001) in SCr fromD0(0.46±0.166 mg/dl) vs. D3(0.54±0.35mg/dl) and D7(0.44±0.22 mg/dl).There was significant difference (p=0.001) in mean levels of serum UA from D0(4.12±2.40 mg/dl) vs. D3(3.82±1.73 mg/dl) and D7(3.56±1.42 mg/dl).Similarly, there was significant difference(p=0.001) in S.P from D0(4.24±1.34 mg/dl) vs. D3(4.61±1.76 mg/dl) and D7(4.13±1.07mg/dl).There was significant difference (0.038) in K from D0(4.10±0.72 meq/L) to D4 (3.92±0.55 meq/L) only. There was no significant difference of mean serum Ca from D0 vs. D3 (0.092) and D7 (0.277).Comparative changes in various biochemical parameters from base line (D0) to D3 and D7 of induction chemotherapy are shown in Over all, TLS was found in 57 (62.6%) children with ALL either spontaneously or after induction chemotherapy. All patients fulfilled the criteria of LTLS whereas clinical TLS was found in 8 (14. 03%) cases. Though, AKI was found in 11 cases but three did not fulfil TLS criteria. However, cardiac arrhythmias and seizures were not observed in any case.Comparative frequency of biochemical and renal abnormalities are shown in TLS occurs in 20- 40% in hematological malignancies and is a serious problem associated with high morbidity and mortality in children with leukemia and lymphoma. So, it is important to recognize risk factors and to initiate preventive strategies.4In our study, TLS was very common in children with ALL and LTLS was found in 62.6%and CTLS in 14% of cases. The reported prevalence varies across the world and our findings are higher than the reported figures, ranging from 25.5 to 39%.19In local studies on various aspects of childhood ALL, the reported frequency of TLS varies from 10-39%.21Considering the biochemical abnormalities in children with TLS, most common abnormality was hyperuricemia (59.3%) followed by hyperphosphatemia (53.8%). Though, our patients were receiving allopurinol along with intensive hydration, but it seems that elimination of circulating uric acid was not enough in the face of rapid production. Second most common biochemical abnormality was hyperphosphatemia. In study by Bagshi et al. hyperphosphatemia was the most common (95%) abnormality and all patients with hyperphosphatemia had AKI and 81% had hyperphosphatemia even before chemotherapy.22Two or more than two metabolic abnormalities were found in 73 %of cases with LTLS whereas in CTLS  more than 87.5% children had two biochemical abnormalities suggesting that all those who developed AKI after cytoreduction therapy was result of hyperuricemia and hyperphosphatemia. Almost all children with high risk stratification (n=56) and pre-B cell leukemia had developed TLS in our study. Similar prevalence of TLS in high risk ALL  and pre-B cell phenotyping has been reported by Mansoor et al and Saeed F et al (37%) recently.23The prevalence of AKI in different studies varies from 5-40% but more recent reports suggest that incidence of AKI varies from14.2-75% depending upon type of patients more in aggressive lymphomas and leukemias. Khalil and colleagues reported AKI in 31.8% in adults with lymphoma whereas Waseem et al. has shown AKI in 40% cases.On comparison of serum UA, P on D0 with D7post-chemotherapy, we found significantly higher than from baseline suggesting that induction chemotherapy is the main risk factor for developing TLS. One most important consideration of our study was that majority (61.5%) of patients had high risk category and pre-B ALL on risk stratification and these patients are more likely to developed TLS due to hyperleukocytosis. In addition, high LDH levels (2499.3±3926.4U/L) in our patients may be an indicator of TLS has also have contributed for development of TLS in this high-risk group.25Tumor Lysis Syndrome was frequent complication in pediatric ALL. Despite of all preventive measures like hyperhydration and use of allopurinol in our study, we found high frequency (62%) of TLS. We suggest more vigilant assessment and monitoring to recognize and treat those patients who are at risk of TLS. Our study’s strength was that it has focused a single and most common childhood malignancy compared to inclusion of multiple hematological malignancies in most studies. Main limitation is retrospective study from single center. A large multicenter study is needed to establish exact incidence in pediatric hematological malignancies.


ТР53 Arg72Pro polymorphic marker with cervical cancer (CC) in Kyrgyz women. The aim of this study was to ascertain the magnitude of association of gene ТР53 gene Arg72Pro polymorphism were identified using PCR-RFLP assay.We identified and included 205 women of Kyrgyz ethnicity for this case-control study, of whom N=103 were women (mean age 53.5 ± 10.0 years) with histologically confirmed CC and N=102 controls (mean age 46.5 ± 8.5 years). We detected human papilloma virus (HPV) DNA types 16 and 18 using polymerase chain reaction (PCR) with hybridization/fluorescent detection. Genotypes of  ТР53 gene showed no difference between CC and control groups . However, when CC cases associated with HPV were tested against controls, Arg72 allele and Arg72Arg genotype prevalence were greater compared to controls . In HPV-positive women, Arg72Arg genotype of ТР53 gene was associated with a 1.85-fold increase in the likelihood of CC  1.03-3.32]), whereas Arg72 allele increased this likelihood 1.94-fold (OR=1.94 [95% CI 1.20-3.15]). Eighty-eight percent (90/103) women with CC had HPV, of whom 43.4% (39/90) had HPV type 16, 24.4% (22/90) had HPV type 18, whereas 32.2% (29/90) carried both types. The univariate analysis of allele and genotype distribution of Arg72Pro polymorphic marker ofТР53 gene in Kyrgyz women increase the risk of HPV-associated CC. Arg72Arg genotype and Arg72 allele of  Cervical cancer (CC) incidence across the globe varied dramatically from somewhat 3.5 to 21 cases per 100 thousand women . In developed countries, this cancer incidence shows the trend for reduction, which may associated with better awareness of risk factors, active prevention and advanced pre-cancer diseases recognition and treatment. On the contrary, in developing world, CC is the leading type of cancer among all malignant tumors in women. Some small, but persistent CC incidence increase is reported in Kyrgyzstan since mid-90s. In Kyrgyzstan, CC incidence in 2011 was 11.0 cases per 100,000, including 60.5% with stage I–II; 30.6% with stage III and 8.9% with stage IV. Mortality rate was 3.3 per 100,000, one-year mortality was 46.9%; and five-year survival was 39.7%. The mean age of CC patients was 40 years . CC prevention is one of prioritized directions in public health and health promotion. CC is to large extent associated with human papilloma virus (HPV). HPV DNA was found in more than 99.5% of biopsy material of CC patients . At present, there exist more than 120 HPV types, whereas 30 of those infect anogenital area. Some HPV types exhibit high oncogenic risk including types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73 and 82. HPV type 16 is considered as most prevalent in CC and found in up to 50-70% CC cases, followed by type 18 (confirmed in 10-20% CC cases) . Vaccination against HPV was initiated in 2007 in countries with efficient public health system. On the contrary, such vaccine is not yet available in low-income countries such as in Kyrgyzstan.Usually it takes ten years and more for malignant transformation from the initial contamination. Viral genome may remain latent for a very long time, and its activation is one of the pivotal stages in oncogenesis, however, this process remains poorly understood. Recent studies have shown that aside from HPV, suppressor genes may also play role in CC pathogenesis. р53 gene is one of the few most studied suppressor genes, important for induction and progression of carcinogenesis associated with HPV . Few studies have confirmed allele status of Р53 gene is associated with CC in HPV-positive women . ТР53 (rs1042522) gene in CC of HPV-positive women in Kyrgyz Republic. This work is the first genetic study of HPV in Kyrgyz Republic. The aim of this study was to assess the role of Arg72Pro polymorphic locus of Study Outline and Sample Population This study was designed as case-control. Cases were 103 CC women, treated in the in-patient department of gynecology of the National Center of Oncology and Hematology in Bishkek from 2015 to 2016. In all included cases the CC diagnosis was based on morphology tests, clinical and ancillary examinations, including ultrasonography and computer tomography (CT), oncogynecologist visit, smear cytology using Papanikolau method and colposcopy. In all cases the diagnosis was verified with biopsy of smears obtained at gynecological consultation and after surgery.One-hundred and two more women were controls, who were unrelated with the women in the main group. Women with the history of any cancer were excluded from controls at a stage of screening. The mean age of CC women was 53.5 ± 10.0 years and 46.5 ± 8.5 years in controls. Only women of Kyrgyz ethnicity constantly residing on the territory of Kyrgyz Republic were included in cases and controls. All participants of both groups signed informed consent to participate. The study protocol was approved by the local institutional review board of the Institute of Molecular Biology and Medicine in Bishkek Kyrgyz Republic .Molecular methods of cervical smears tests Epithelial cell smears from the cervix were the material for molecular tests in this study. High carcinogenic risk HPV type 16 and 18 were detected and differentiated using reagents set «AmpliSense, HPV 16/18-FRTA» via PCR with hybridization/fluorescent detection in real time. This method is based on a simultaneous HPV DNA segments amplification along with the segment of β-globin gene DNA, using for the purpose of endogenous internal control. All test stages, such as nucleic acid separation, amplification, analysis and interpretation, were completed as of the producer’s manual (High-Risk Human Papilloma virus Infections). DNA extractionDNA was extracted from the white cells of peripheral blood, using the standard method of phenol-chloroform extraction. Determination of ТР53 Genotype ТР53 gene using PCR with subsequent restriction of its products and restriction fragments length polymorphism (PCR-RFLT) assay. PCR was conducted in Hybaid (UK) amplifier. We amplified ТР53 gene Arg72Pro locus with direct 5`TTGCCGTCCCAAGCAATGGATGA–3` and reverse 5`TCTGGGAAGGGACAGAAGATGAC–3` primers. Twenty µL of amplification mixture contained 1x buffer for Go-Taq-polymerase, 1.5 мМ MgCl2, 1.6 мМ of dNTP mixture, 5 pmol of each primer, 100 nanogram of genome DNA and 1 unit of Go-Taq- polymerase . Amplification protocol included preliminary denaturation at 94°С for 5 minutes with 35 subsequent cycles of denaturation at 94°С (30 sec), annealing at 60°С (30 sec) and chain elongation at 72°С for 30 sec. The program was finalized with elongation at 72°С for 5 min. Amplification products were then hydrolyzed with BstUI endonuclease  at 37о for 16 hours. Restriction products were separated usng elecroforesis in 3% agarose gel and the fragments were visualized by means of GelDoc IT system  in ultraviolet light. Arg72 allele has fragments 133 and 86 b.p. long, where as 72Pro minor allele is 199 b.p. long . Differences were considered significant with р<0.05.We used previous report from Chinese population to calculate the current study sample size using the built-in formula in Equation 6.10. In the preceding study , the odds ratio (OR) of CC in Arg72Pro carriers was 3.83. Given the number of exposed controls in our study 35%, case:control ratio 1:1 and Z-alpha 1.96, the minimum required number of cases for OR 3.83 should be 36 with the corresponding number of controls 36 also to attain 80% power. Taking stricter OR 2.38 for Pro72Pro from the same study, the minimum required number of cases would be 86. Therefore, our study sample size of 102 cases ensures sufficient study power (100% and 84% correspondingly). We analyzed data in GraphPad Prism v 5.04. We tested the observed distributions vs. the expected Hardy-Weinberg equilibrium via χSubjects’ profileAt the time of diagnosis, the age of CC patients ranged from 20 to 77 years. Forty-five women (44%) in the CC group (N=103) were in the age range from 51 to 60 years, whereas younger women with the age range 41 to 50 years were less prevalent . There were very patients in the age group of 20 to 30 years , 15 patients (14.5%) were 31-40 лет years old, 9 women (8.9%) were 61-70 years old and only 5 patients (5%) were older than 70. Among the range of existing CC stage classifications, we used FIGO scale to grade patients. In the current sample, clinical stage Ι was found in 3 patients (3%), stage ΙΙ in 35 patients (34%), stage ΙΙΙ was identified in 62 cases (60%), and stage ΙV was found in 3 (3%) patients. Squamous cancer was diagnosed in 98 (95%) women, whereas adenocarcinoma was found in 5 (5%) patients. G2 tumor was verified in 100 (97%) included women, whereas G3 in 3 (3%).Ninety women (87%) of the entire pool of 103 CC patients were infected with HPV, of whom 43.4% (39/90) had HPV type 16; 24.4% (22/90) had type 18 and 32.2% (29/90) exhibited HPV of both types (16/18). HPV was found in one subject of the control group. Distribution of genotypes and allele frequencies among subjectsТР53 Arg72Pro polymorphic locus in CC women and in control group reflected Hardy-Weinberg equilibrium . In both group we found that Arg allele and Arg72Arg genotype prevailed over the alternative variants. Arg72Arg, Arg72Pro and Pro72Pro genotype prevalence in the group of women with CC were 58%, 33% and 9% correspondingly, whereas Arg and Pro alleles’ frequency was 0.74 and 0.26.Genotype distribution of gene 2 = 1.24, р=0.54).The corresponding frequency of Arg72Arg, Arg72Pro and Pro72Pro genotypes in the control group was 52%, 35% and 13%, whereas Arg and Pro allele’ frequency was 0.70 and 0.30. We did not find significant differences in the prevalence of genotypes and alleles of gene Р53 Arg72Pro polymorphic locus when comparing CC patients with controls  . In HPV-The association of CC stage and differentiation with the genotypes of TP53 gene Arg72Pro polymorphic locus Patients were divided into three groups as of their genotypes. The first group consisted of patients with Arg72Arg homozygous genotype; the second group was formed from Arg72Pro heterozygous genotype; finally, Pro72Pro minor genotype made the third group .The association of genotype with tumor malignancy (G)In our sample, more than half of all CC had II degree of differentiation. Moderate differentiation (G2) was present in 97% cases (n=100), whereas low differentiation (G3) was identified in 3% (n=3) . As TablThe association of genotype with the disease stage (FIGO)We also analyzed the distribution of tumor stages in a sample of CC women. Clinical stage Ι was found in 3 patient (3%), stage ΙΙ in 35 patients (34%), stage ΙΙΙ was identified in 62 cases (60%), and stage ΙV was found in 3 (3%) patients. In a comparative analysis, we could not associate the stage with alleles or genotypes of P53 gene’s Arg72Pro polymorphic locus .The association of genotype with tumor histological type We also tested the association of genotypes of TP53 gene’s Arg72Pro polymorphic locus with tumor histological types. Histological examination revealed squamous cancer in 97% women with CC, whereas adenocarcinoma was found in 3%. There was no statistically significant difference in the distribution of squamous cancer or adenocarcinoma in various genotype combinations  .ТР53 gene with CC in HPV-positive women of Kyrgyz ethnicity. CC is believed to be a HPV-associated disease , and in our study, 87% (90 out of 103) women with CC had confirmed HPV. Our analysis showed that CC risk in HPV-positive women was associated with Arg allele carriage  and Arg/Arg genotype . On the contrary, Pro allele was protective against CC , as was Pro/Pro genotype . Our data also confirm the association of Pro72Arg marker of ТР53 gene is located on a short shoulder of the 17th chromosome (17p13.1) , contains 11 exons and transcribes 2.8 kbmRNA. Phosphopotein р53 is a protein product of the gene and contains 393 amino acids. р53 protein regulates cell cycle, induces apoptosis in case DNA impairment cannot be repaired, and stabilizes genome. Cell cycle regulation impairment, which is caused by р53 activity change, will promote cancer progression. Such defect can be caused both by mutations and polymorphism .ТР53 has around 200 known single nucleotide polymorphisms (SNP) [http://www-p53.iarc.fr/]. Guanine replacement with cytosine in position 215 of the fourth exon (Arg72Pro (rs1042522)) is considered one of the most meaningful polymorphisms of ТР53 gene. Such mononucleotide replacement results in two versions of р53 protein, including the one with arginine (Arg) and one with proline (Pro) in the 72nd codon . Biochemical properties of these two protein variants differ. Data from the literature show that 72Arg ТР53 protein may have a greater potency to initiate apoptosis compared to 72Pro ТР53, because 72Arg protein penetrates mitochondria quicker, and that entails cytochrome C release into the cell cytoplasm and subsequent apoptosis induction .ТР53 gene may promote CC, associated with papilloma virus. They also reported that Е6 oncoprotein, coded by HPV-18 and HPV-16, may interact with р53 protein, and that induces its degradation . Storey et al.  found that p53Arg allele is destroyed by Е6 HPV-18 much faster than p53Pro. Mutant variant of this protein loses this property, and that leads to cancer transformation. The fact that all cancer cells exhibit the mutant variant of р53 inclined the researchers to conclude that the normal variant of this protein may act as a carcinogenesis suppressor. Based on that, the authors hypothesized that allele status of Storey et al., (1998) found a 7-fold increase of CC risk in HPV-positive women with Arg72Arg genotype. Min-min et al., (2006) also found higher CC risk in HPV-positive Chinese women with Arg72Arg genotype when compared to their Pro72Pro counterparts. Other studies in India , South Africa  and Santiago  also found greater CC risk in HPV-positive women with Arg72Arg genotype. Population-based studies in the European region, including Italy , Lithuania  and Germany  identified similar association of higher CC risk in HPV-positive women with Arg72Arg genotype when compared to two other variants. On the contrary, other authors reported no association of Arg72Pro polymorphic locus with CC risk in HPV-positive women . Our findings are quite consistent with a number of other studies showing higher susceptibility of HPV-positive women to CC when having Arg72Arg genotype and 72Arg allele. ТР53 gene Arg72Arg polymorphism variants may depend on the geographical location. Thus, Arg72 allele prevalence in the Irish was 0.88; 0.72 in Italians; 0.63 in Germans; 0.65 in Russians; 0.52 in our sample of Kyrgyz; 0.42 in Koreans; 0.44 in Japanese; 0.34 in Jews; 0.33 in South Africans and 0.27 in Indians . In other words, Arg72 allele prevalence lessens closer to the equator. This may be important to verify the distribution and frequency of selected neoplasms associated with HPV 16 and 18 in the given population. Of note, the prevalence of Ethnicity is an important contributor to allele distribution in the general population since numerous studies have shown different prevalence of specific alleles, including mutations, between ethnic groups. Therefore, selection of ethnically homogenous groups is pivotal for molecular and genetic studies. In this analysis, we report genotyping data of CC patients along with controls from one laboratory and using standardized methodology. The groups were compiled from one population, which prevents inter-population variations.The strength of this report is the first presentation of the association of Arg72Pro polymorphic locus with CC in HPV-positive women in Kyrgyz population. The limitations of this study are small sample size, inclusion of only one gene for the polymorphic locus evaluation.In conclusions, given the high prevalence of papilloma viral infection among women combined with HPV etiology in CC, identification of the population with high genetic risk of CC is of significant importance. This will allow reducing such risk, since timely vaccination against HPV will be targeted. The authors declare no conflicts of interest. This study protocol was approved by the local committee on bioethics of the Institute of Molecular Biology and Medicine , Bishkek, Kyrgyz Republic.


The study period was from 2012 to 2016. Anemia was defined according to the World Health Organization (Hb < 11 g/dL). PM2.5 values were estimated by a mathematical model that combined data observed from monitors, with satellite and meteorological data. PM2.5 was analyzed by quintiles. Multiple linear and logistic regressions were used to estimate the associations between hemoglobin concentration (beta) and anemia (odds ratio) with PM2.5, after adjusting by covariates. Prevalence of anemia was 39.6% : 39.3–39.9). Mild anemia was observed in 30.8% of children and moderate/severe in 8.84% of children. Anemic children compared with nonanemic children are mainly males, have low body weight, higher rate of stunting, and live in an environment with high PM2.5 concentration. A slight decrease in hemoglobin  and an increase in the probability of moderate/severe anemia  were observed with increased exposure to PM2.5. We conclude that outdoor PM2.5 levels were significantly associated with decreased hemoglobin values and an increase in prevalence of moderate/severe anemia in children under 5 years old.Anemia affects 1.62 billion people worldwide. Although iron deficiency is the main cause of anemia, several other factors may explain its high prevalence. In this study, we sought to analyze the association between outdoor particulate matter PM Anemia affects 1.62 billion people worldwide [According to the WHO, 50% of cases of anemia are due to iron deficiency, while 42% of the cases are of inflammatory origin (inflammatory anemia). Inflammatory anemia or anemia of inflammation (AI) in children aged 6–59 months is mainly related to infectious diseases particularly in less developed countries . However2.5) and NO2 produce systemic inflammation [2.5 is one of the most toxic pollutants, and short-term exposure to PM2.5 causes a 2.8% increase in mortality rates with adverse effects in human health [2.5 is associated with cardiovascular disease and chronic obstructive pulmonary disease [Another potential cause of inflammatory anemia is exposure to air pollutants since fine particulate matter equal or less than 2.5 microns in diameter  and anemia is also observed in other age groups and how associated it is to the severity of anemia. A study in 29 developing countries revealed that both moderate exposure and high exposure to biofuel smoke at the country level were associated with moderate/severe anemia in children (odds ratio (OR): 2.36; 95% confidence interval (95% CI): 1.28–4.36 vs OR: 2.80; 95% CI: 1.37–5.72) .A strong association between biofuel use and risk of anemia and stunting in children suggests that exposure to biofuel smoke may contribute to chronic nutritional deficiencies in infants. 31% of moderate-to-severe anemia and 37% of severe stunting among children aged 6–35 months in India may be attributable to exposure to biofuel smoke .2.5, on anemia in children is still unknown.Despite there are data regarding indoor air pollution and anemia, the effect of outdoor air pollution, especially PM2.5 values, a matter of severe public health concern [2.5 levels in Lima between 2001 and 2011 averaged 50 μg/m3. It was estimated that the excess of PM2.5 in Lima (above 10 μg/m3 of the reference level) during this period, resulted in approximately 2,300 premature adult deaths from cardiorespiratory disease annually [According the World Health Organization (WHO), Lima, Peru, is one of the Latin American cities with the highest mean annual PM concern . In factannually .2.5 concentration should be 10 μg/m3 and 25 μg/m3 for a 24-hour mean [μg/m3 for an annual mean and 50 μg/m3 for a 24-hour mean [Air quality guidelines given by the WHO state that the annual mean value of PMour mean . This thour mean . These d2.5 concentration in children aged 6–59 months residing in Lima, Peru. The study also aims to determine the association between anemia levels and high PM2.5 values using the thresholds provided by the WHO and MINAM.For the aforementioned reasons, the present study was designed to determine the relationship between Hb and anemia with PM2 of the land; those include the actual city (825.88 km2) and the city outskirts . For this study, districts of Lima have been divided into 5 zones: North Lima, Center Lima, South Lima, East Lima, and West Lima . Center Lima is considered more economically developed than the rest of the zones, but the entire city is highly urbanized. The population density of Lima by district can be seen in Supplementary The metropolitan city of Lima is the capital of Peru with more than 10 million inhabitants. It is located in the central coast of the country (150 m.a.s.l.), next to the Pacific Ocean. Lima is divided into 43 districts, which cover 2,672.28 kmn = 28) or >30 g/dL (n = 64). These data were excluded from analysis. Data were collected yearly from 2012 to 2016.The study analyzed the Nutritional Status Information System (SIEN in Spanish), a database provided by the National Center of Feeding and Nutrition (CENAN in Spanish), an institution belonging to the National Institutes of Health in Peru. Data analyzed in the current study included 139,368 infants and children aged 6 to 59 months with permanent residence on the districts that comprise the metropolitan city of Lima. Distribution of the sample by district can be seen in Supplementary Hemoglobin (Hb) was measured in each health center by trained personnel using the Hemocue method and is expressed in grams per deciliter (g/dL). Anemia in children aged 6 to 59 months was defined as Hb < 11 g/dL according to the WHO guidelines . Anemia 2.5 exposure levels from 2010 to 2016 were estimated using a random forest (RF) model [https://aeronet.gsfc.nasa.gov/cgi-bin/type_one_station_opera_v2_new?site=Arica&nachal=2&level=1&place_code=10), the nearest AERONET site located in Chile for 2010 to 2015, and gap-filled through a random forest model  R2 = 0.82) [Daily PMF) model . The RF  = 0.82) .2 and 1 km [2 resolution through inverse-distance weighting. Land use variables such as percent urbanization, elevation, population density, and distance to road were also calculated for use in the model. The RF model to predict daily PM2.5 levels achieved an R2 (and CV R2) of 0.70  [Meteorological variables from WRF-Chem (Weather Research and Forecast model coupled with Chemistry) and ECMWF (European Centre for Medium-Range Weather Forecasts) were collected at 5 km7 μg/m3) .2.5 data was estimated from each participant's district of residence.Finally, we assigned the average of the last month prior to the measurement of hemoglobin to each child. PMOther variables were age (expressed in months) and stunting (defined as height for age Z-score (HAZ) < −2.0). Lima area includes the 43 districts grouped as north (8 districts), center (15 districts), south (12 districts), east (8 districts), and west  (Supplementary This study was approved by the IRB at the Universidad Peruana Cayetano Heredia (SIDISI 103700).http://www.qgis.org) was used for map construction. Data are presented as mean values ± standard deviations for continuous variables and as percentages for categorical variables. For statistical analysis, anemia was further categorized as mild and moderate/severe since cases of severe anemia were scarce. PM2.5 concentration does not have normal distribution; therefore, nonparametric tests were used to compare PM2.5 and anemia in the different zones of Lima. For categorical variables such as anemia and stunting, the chi-square test was used to compare them between the five zones of Lima. Differences between normally distributed variables among the five zones of Lima were assessed with ANOVA test. Those variables that were not normally distributed were assessed with nonparametric Wilcoxon test.Software STATA 14.0  was used for statistical analysis and QGis 2.18.16 Las Palmas ; 2Q: 19.18 μg/m3 (18.23–20.25); 3Q: 21.89 μg/m3 (20.26–24.89); 4Q: 26.87 μg/m3 (24.97–28.83); and 5Q: 32.69 μg/m3 (28.85–51.0). We additionally generated a new variable with values recommended by the WHO (25 μg/m3) and MINAM (50 μg/m3) as cut-off of permissible PM2.5 values in order to compare the risk between both thresholds.Multiple linear regression and logistic regression models were used to associate hemoglobin concentration or prevalence of anemia with PMp value <0.05 was considered as significant.To assess if a certain age group is more vulnerable to the exposure, we further stratified by age from 6 to 11, 12 to 35, and 36 to 59 months. This categorized variable has been included in all statistical models. A n = 139,368 children), 55,216 (39.6%) children had anemia (Hb < 11 g/dL). Anemic children compared to nonanemic children are mainly young children, male, with low body weight, higher rate of stunting, and live in an environment with high PM2.5 concentration. Mild anemia was observed in 30% of the population, while severe/moderate anemia was observed in 8.8% of the population.Characteristics of the study population are shown in 2.5 (31.6 μg/m3 ± 5.6). The highest concentration of Hb was observed in the West zone, and it was associated with the lowest prevalence of anemia, lower prevalence of stunting, and lower PM2.5 values.In 2.5 in the districts of metropolitan Lima from 2012 to 2016. The lighter blue tone corresponds to lower concentration of PM2.5 (13.6–18.23), and the darkest tone corresponds to the highest concentration of PM2.5 (28.85–50.83). 2.5 concentration and hemoglobin showed a decline in Hb concentration with increasing PM2.5. Interestingly, exposure to values of the quintile 4 (24.97–28.84 μg/m3) resulted in a significant decrease in hemoglobin levels .When magnitude of anemia was assessed, the effect of exposure to high concentrations of PMuintiles . Quintil2.5 permissible values and their association with anemia. When using the threshold recommended by the WHO (PM2.5 = 25 μg/m3), a reduction of hemoglobin is observed even at lower than permitted values . With the threshold recommended by the MINAM (PM2.5 = 50 μg/m3), a greater decline of hemoglobin concentration was observed .We analyzed the thresholds used by the WHO and MINAM as cut-off point to define PM2.5 was associated with moderate/severe anemia . However, when using MINAM reference values, PM2.5 over the threshold were associated with both moderate/severe anemia  and mild anemia .Mild anemia and severe/moderate anemia have been evaluated at the different air quality cut-off points. When using the threshold recommended by the WHO, PM2.5 was negatively associated with hemoglobin levels at 6–35 months of age . This was not observed in the group of 36–59 months (p=0.597).After adjusting for confounding variables, such as age, gender, stunting, season, poverty, month, year, and Lima zone, PM2.5 exposure and its association with anemia prevalence in children aged 6 to 59 months residents in Lima, Peru, one of the cities with the worst air pollution in Latin America. The anemia rates shown in these studies are high and are in line with official statistics for metropolitan Lima [2.5 is associated with a decrease in hemoglobin levels. This, in turn, increases the prevalence of anemia. For instance, when people are exposed to concentrations between 25 μg/m3  and 50 μg/m3 , a decrease in hemoglobin is observed. Moreover, the higher the concentration of PM2.5 to which a child is exposed, the greater the reduction in hemoglobin concentration.This study assessed outdoor PMtan Lima . Anemia 2.5 that differs from the ones recommended by the WHO. The prevalence of anemia increases at 50 μg/m3 PM2.5 [2.5 (25–50 μg/m3), meaning that particulate matter at higher concentrations is an important health problem for populations exposed to this pollutant.Peru has its own guideline thresholds for PMm3 PM2.5 . Also, a2.5 values below the threshold recommended by the WHO. Most of the children with anemia have mild anemia [2.5) moving anemia from mild to moderate/severe. Our hypothesis is based on the fact that threshold of PM2.5 recommended by the WHO and MINAM was associated to moderate/severe anemia.Furthermore, our results report an association with moderate/severe anemia, even with PMd anemia . The gro2.5 hold a greater risk of a decrease in hemoglobin values and an increase in anemia prevalence. With MINAM's referential daily value of 50 μg/m3, children are at an even higher risk for moderate/severe anemia. Data from the present study reveal that even values below the threshold recommended by the WHO are associated with anemia in children. Guidelines to define permissible values of daily PM2.5 concentration should be revised. In Lima, the main sources of outdoor air pollution are automotive fleet and industrial activities [2.5 values [As evidenced by the results in this study, children exposed to high concentrations of PMtivities . A recen5 values , meaningIn Peru, children begin preschool after turning 3 years old. Before this age, they mainly stay at their homes, so assessing the exposure with the district of residence gives a good estimate of how permanently exposed these children are to pollutants. Furthermore, children tend to attend preschool institutions close to their homes at the same district, so estimating exposure based on the district of residence is also applicable for the older age group.2.5 has been shown to increase systemic inflammation [10 were associated with a reduction of hemoglobin concentration and red blood cell counts but increased white blood cell counts suggesting a direct association with inflammation and an inverse relationship with hemoglobin levels [2.5 was associated with a decrease of 0.81 g/dL of hemoglobin in an elderly population [The plausibility of our findings is that air pollutants generate an inflammatory process in the human body , 27. PM2ammation  and prodammation . These sn levels . Furtherpulation . Other vpulation .2.5, C-reactive protein levels, an inflammatory marker, increase [2.5 exposure and anemia.Particulate matter, as any other pollutant, generates an inflammatory response in the human body –35. Thesincrease . Therefo2.5 increases serum hepcidin levels, degrading ferroportin as a host defense mechanism [2.5 on hepcidin and the hemolysis need further research.Hepcidin, the hepatic peptide hormone in charge of controlling iron homeostasis, and ferroportin, a transmembrane receptor which exports iron into blood plasma, control the main routes of iron transport and availability and determine total body iron content . Inflammechanism . This meechanism . Anotherechanism . Both thThis study has certain limitations. First, our database was obtained from the Information System of Nutritional Status (SIEN in Spanish) of the National Institutes of Health in Peru. The rates of anemia prevalence only correspond to the population which was attended at public health care centers. People attended in private health care system, which accounts for 10% of the population, and those attended by the Social Health Insurance , nearly 39% , are not2.5 data have been linked to each participant's district of residence; it could not be linked to each participant's exact address. Another major limitation is that inflammatory markers have not been assessed in this study. C-reactive protein (CRP) and hepcidin levels should be measured in future studies to properly explain the mechanism by which exposure to increased PM2.5 increases anemia prevalence.This limitation may explain why some of the more residential or high-income districts have a high percentage of anemia prevalence since it does not include the populations with a high or high-average socioeconomic level that are attended at private health care centers. PM2.5 concentrations, particularly over 50 μg/m3. This, however, needs to be proven in further research, as well as the higher susceptibility of younger children since PM2.5 is more strongly related to anemia in the younger age group.Even though the study is not prospective, it appears as children with mild anemia (control group) may worsen and become moderate to severe anemic when exposed to increased PMμg/m3) concentrations of PM2.5 is associated with an increased prevalence of anemia in children aged less than five years particularly moderate/severe anemia, constituting a new risk factor to be taken into account when designing policies aiming to eradicate anemia worldwide. Since the risk of decreased hemoglobin levels is seen even at lower values than those suggested by the WHO and local regulatory agencies (MINAM), we call for a revision of current guidelines. The mechanisms by which PM2.5 exposure might decrease hemoglobin concentrations need to be studied further but might be related to the activation of inflammatory processes.Anemia is currently the major public health problem Peru is facing; this paper sheds light on other reasons that might explain the high anemia prevalence in the country. Exposure to higher than allowed (25


Introduction: Burn wounds are commonly infected by organisms which delay wound healing. Therefore, it is necessary to evaluate the flora obtained from wounds of burn patients in order to determine the most effective treatment. The aim of this study was to determine the frequencies of various bacteria isolated from burn wounds and to determine their antimicrobial susceptibility. Materials and methods: This descriptive cross-sectional study was conducted from January 2018 to November 2018 which included consecutive samples of burn wounds from patients admitted to the burn ward of a tertiary care hospital. Bacteria and their antimicrobial susceptibility were determined by swab cultures and sensitivity tests by standard aseptic techniques. Data were analysed via the Statistical Package for Social Sciences (SPSS), v23.0 . Chi-square tests were applied between qualitative variables, while the Kruskal-Wallis test was applied to compare the means of asymmetrical data. Bacterial isolates and their susceptibility pattern were represented as frequencies and pie charts.Pseudomonas aeruginosa - 41 specimens (24.91%), followed by Staphylococcus aureus - 38 specimens (24.05%), Acinetobacter - 27 (17.09%), Klebsiella - 24 (15.19%), Escherichia coli - 13 (8.23%), Proteus - 7 (4.43%), other coliforms - 6 (3.8%), Enterococcus - 1 (0.63%), and Enterobacter - 1 (0.63%). Drug resistance to penicillin G, ampicillin, Augmentin, ceftazidime, cefotaxime, ceftriaxone, meropenem, and piperacillin+tazobactam was exceptionally high.Results: A total of 178 samples were obtained from 109 patients from burn wounds. One hundred and twenty-two wounds (68.5%) showed growth and 56 (31.4%) showed no growth after 24 hours of incubation. Positive cultures were significantly more frequent in wounds of greater than one-week duration (p < 0.002). Out of 158 bacterial isolates, the most common isolate was Pseudomonas aeruginosa and Staphylococcus aureus. Piperacillin+tazobactam against Pseudomonas aeruginosa and vancomycin and linezolid against Staphylococcus aureus are highly effective and can be used as empirical therapies.Conclusion: The most common bacterial isolates are  Burn injuries to the skin and other tissues are caused by heat, radiation, electricity, friction, or contact with chemicals . Around Burn wounds are a susceptible site for the growth of opportunistic organisms of endogenous and exogenous origin . InfectiAn increase in the patient load in government hospitals causes a delay of a few days between ordering the cultures and receiving culture reports. During this crucial time, empirical antibiotic therapy can be given to the patients to control the infection. However, due to the inappropriate prescription of antibiotics  and theiThis descriptive cross-sectional study was conducted in the burn unit of a tertiary care hospital. The Institutional Research Forum, Rawalpindi Medical University issued approval RSRS-2018-P-01. The study was conducted from January 2018 to November 2018 using consecutive non-random sampling. We included male and female patients of all ages who were admitted to the burn unit of our hospital. Patients who were admitted again after being discharged once, either for follow-up or for plastic surgery procedures, were excluded. One hundred and seventy-eight culture reports of 109 patients were included. Patients who had a prolonged hospital stay had multiple culture reports, while those who had minor burn injuries and short hospital stays had only one culture report at the time of hospital admission. All patients had their wound cultures and sensitivities done at the time of admission. Samples were collected from burn wounds by culture swabs under aseptic techniques. They were then sent to microbiology laboratory where samples were inoculated on agar plates. Wound culture was done on blood and McConkey agar. Cultures were incubated at 37°C for 24 - 48 hours. Microbes were identified under the microscope by observing morphological characteristics and applying biochemical tests. Antibiotic sensitivity pattern was done on Muller Hinton agar by Kirby Bauer disk diffusion method. Age, gender, total body surface area burnt (TBSA), type of burn, and date of admission were collected from the patients' record.Data was entered and analysed using the Statistical Package for Social Sciences (SPSS), v23.0 . Descriptive statistics were applied to find frequencies, percentages, means, and standard deviations. Quantitative variables, such as age and TBSA, were expressed as means and standard deviations. Qualitative variables, such as type of bacteria and type of burn injuries, were expressed as frequencies and percentages. Normality of quantitative variables was checked by applying Kolmogorov-Smirnov and Shapiro-Wilk tests. Independent samples Mann-Whitney U test and Kruskal Wallis test were applied to find a significant difference between means of non-parametric variables having two and more than two categories, respectively. Chi-square tests were applied to compare the percentages of two categorical variables. Level of significance was set at p < 0.05.A total of 178 culture reports from 109 patients. Sixty patients (55%) were males, while 49 patients (45%) were females. Ages ranged from two years to 75 years. The median age was 21 years (interquartile range (IQR) = 25.75). The percentage of burns ranged from 3% to 95%. The median TBSA was 30% (IQR = 23.50). Most of the burns encountered were due flame - 70 (64.2%), followed by electric burns - 20 (18.3%), scald - 16 (14.7%), acids - 1 (0.9%), and alkalis - 1 (0.9%). The type of burn was missing for one patient (0.9%). Flame burns were significantly more frequent in females (52.9%) than in males (47.1%), while scalding and electric burns were significantly frequent in males (56.3% and 85%) than in females  (p < 0.03). The results of independent samples Kruskal-Wallis test showed that the distribution of age was significantly different among the type of burn injury. Scald burns were significantly more frequent in children, while flame and electric burns were more frequent in adults (p < 0.000). Out of 178 culture reports, 122 (68.5%) showed positive growth while the remaining 56 (31.5%) showed negative growth. Positive growth was significantly more frequent after the first week of hospital stay (p < 0.002). Positive growth was also more frequent in burn injuries due to causes other than flame (78.9%) than in flame injuries (61%) (p = 0.012). Out of 122 positive growths, 88 (49.4%) had a single growth while 34 (19.1%) showed polymicrobial growth. A total of 158 bacterial isolates were recorded from 122 positive growths  - 41 (24.95%), followed by Staphylococcus aureus (S. aureus) - 38 (24.05%). In third place was Acinetobacter - 27 (17.09%), followed by Klebsiella - 24 (15.19%), Escherichia coli (E. coli) - 13 (8.23%), Proteus - 7 (4.43%), other - 6 (3.8%), Enterococcus - 1 (0.63%), and Enterobacter - 1 (0.63%). Twenty-seven out of 38 (71.05%) S. aureus isolates were methicillin-resistant S. aureus (MRSA). Five out of 41 (12.2%) of the P. aeruginosa were pan-resistant. The pattern of growth is shown in Figure Out of these, 119 (75.31%) bacterial isolates were gram-negative bacilli, while 39 24.68%) were gram-positive cocci. The most frequent bacterial isolate was 4.68% werThe antimicrobial sensitivity pattern of bacterial isolates is shown in Table 5 of tissue in the wound, we call it a wound infection. When more than 105 of tissue in the burn wound causes the formation of pus and separation of the eschar, loss of graft with the involvement of tissue, or the presence of systemic sepsis, then it is called invasive infection. Cellulitis is erythema, induration, warmth, and tenderness in the tissue surrounding the burn wound, and when invasive infection involves the deeper structures below the skin, then it becomes a necrotizing infection [The skin is a protective physical barrier against microorganisms which are disrupted in burn injuries. The disruption of the vasculature below the skin also plays an important role in the growth of microorganisms in burn wounds . Accordinfection , 15.In our study, more males (55%) were affected by burn injuries as compared to females (45%). This is in accordance with the study conducted at the Burn Care Centre of the Pakistan Institute of Medical Sciences (PIMS), Islamabad by Saaiq et al. where 53.68% were males . HoweverWe found that 68.5% of the cultures showed positive growth. This was in contrast with the higher rate of infection in the studies conducted in India where 88.2% and 93% of the samples showed positive growth -18. PosiP. aeruginosa was the most common pathogen isolated from burn injuries. This was inconsistent with many studies [Acinetobacter was the most common organism infecting the burn wounds [S. aureus was the most common organism according to other studies by Altoparlak et al. and Erol et al. [P. aeruginosa was alarmingly high. Sensitivity to piperacillin and tazobactam in our study was 38.9% in contrast to 80.55% in the study by Saaiq et al. [P. aeruginosa showed the highest sensitivity to ceftazidime and meropenem with 55.6% isolates sensitive to each drug [P. aeruginosa to ceftazidime was only 8.8% and sensitivity to meropenem was 19%. After piperacillin and tazobactam, P. aeruginosa showed the most sensitivity to amikacin (25%) in our study. This clearly demonstrates that P. aeruginosa has become more resistant to drugs that were previously thought effective in the literature. Such a low sensitivity of P. aeruginosa demands the introduction of newer antibiotics or other methods of infection control against this organism.In our study,  studies -23, incl studies -25. In cn wounds , whereasl et al. -28. In oq et al.  and 11.1q et al. . In the ach drug . In our S. aureus was the second most common organism contaminating the burn wounds in our setting; however, in another study in India by Datta et al., S. aureus was the most common pathogen [S. aureus was the second most common organism in the burn wounds [S. aureus were methicillin-resistant. This is an approximation to the study by Saaiq et al. where 68.62% of cultures were methicillin-resistant [S. aureus infections were resistant to methicillin in the study by Datta et al. [pathogen . Our finn wounds . In our esistant , whereasa et al. . Gram-poa et al. , 29. MRSa et al. . This shAcinetobacter species were the third most common microorganisms that contaminated the burn wounds in our burn ward. This is in contrast to the study by Saaiq et al. where the third most common pathogen was S. aureus [Staphylococci in the study by Datta et al. [Klebsiella in other studies conducted in India [Acinetobacter in our study with 96% of the samples showing sensitivity to it. It showed very low sensitivity to many other the third generation cephalosporins. This is similar to the study by Hegde et al. where Acinetobacter showed very high resistance to all other drugs, except tigecycline for which 97.5% of the isolates were sensitive [The . aureus  compareda et al. , and Klein India -19. Tigeensitive .Klebsiella species were the fourth most common isolate in our setting which comprised 15.19% of the isolates, whereas in the study by Saaiq et al. Klebsiella was the second most common bacterial isolate [Klebsiella species in our study were most sensitive to imipenem with 44.4% isolates sensitive to it; however, in the study by Saaiq et al., piperacillin and tazobactam were found to be highly effective against Klebsiella with 80.95% sensitive isolates [Klebsiella species were sensitive to piperacillin and tazobactam.The  isolate . Klebsieisolates . In our E. coli to be the second most common pathogen in burn wounds [E. coli in our setting was the most sensitive to imipenem with 66.7% of the isolates sensitive to it, whereas amikacin and meropenem were highly effective against the E. coli in the Lakshmi et al. study [A study in India conducted by Lakshmi et al. found n wounds . Howeverl. study .Proteus species (4.43%), coliforms (3.8%), Enterococcus (0.63%), and Enterobacter (0.63%). In the study by Datta et al. [Proteus, whereas 17.4% had Proteus in the study by Lakshmi et al. [Enterococcus in the study conducted by Hegde et al. was 4% [Enterobacter made up 0.93% of all the isolates, which was similar to 0.63% of Enterobacter isolates in our study [The least common pathogens in our study were the a et al. , 3.33% oi et al. . The per. was 4% . In a stur study .Pseudomonas and Klebsiella was the most common bacterial combination in our study (20.59%); however, in the study by Rajbahak et al., Pseudomonas occurred most frequently with Acinetobacter (12.9%) [ (12.9%) .Our study shows that resistance of all bacteria against penicillins and the third generation cephalosporins has reached a very high level.P. aeruginosa and S. aureus. Piperacillin and tazobactam against P. aeruginosa and vancomycin and linezolid against S.aureus are highly effective and can be used as empirical therapies. The increasing antibiotic resistance of P. aeruginosa against the known antibiotics demands rigorous infection control, discouraging overuse and misuse of antibiotics, the invention of newer and more effective antibiotics, or adopting newer ways to control bacterial infections.Flame burns are the most common cause of burn in females, while scald burns are more common in children. Antibiotic sensitivity pattern is everchanging. The most common bacterial isolates found in the burn wounds are


It may be concluded that sulforaphene, but not its parent glucosinolate, glucoraphenin, causes cytotoxicity and stimulates apoptosis in HepG2 cells.Glucoraphenin, a glucosinolate present in large quantities in radish is hydrolysed by myrosinase to form the isothiocyanate sulforaphene, which is believed to be responsible for its chemopreventive activity; however, the underlying mechanisms of action have not been investigated, particularly in human cell lines. The aim of the study is to assess the cytotoxicity of sulforaphene in HepG2 cells and evaluate its potential to enhance apoptosis. The cytotoxicity of sulforaphene in HepG2 cells was carried out ensuing an initial screening with two other cell lines, MFC-7 and HT-29, where sulforaphene displayed highest toxicity in HepG2 cells following incubation at 24, 48 and 72 h. In contrast, the intact glucosinolate showed no cytotoxicity. Morphological studies indicated that sulforaphene stimulated apoptosis as exemplified by cell shrinkage, blebbing, chromatin condensation, and nuclear fragmentation. The Annexin V assay revealed significant increases in apoptosis and the same treatment increased the activity of caspases -3/7 and -9, whereas a decline in caspase-8 was observed. Impairment of cell proliferation was indicated by cell cycle arrest at the Sub G Cancer is one of the leading causes of mortality throughout the world today. Being an extremely complex disease, treating cancer has become a major challenge. There are more than 200 types of cancers, one of which is hepatocellular carcinoma, reported to be amongst the most typically diagnosed cancers ,2,3.A number of phytochemicals have been considered as potential cancer chemopreventive agents, e.g., glucosinolates (GLs) , major cRaphanus sativus L. It was reported that GRE and another GL, glucoraphasatin, are among the most promising GLs due to them both bearing an extra sulphur function in their aglycon -2,5-diphenyltetrazolium bromide (MTT) assay was performed according to the method by Mosmann . For theTM colorimetric apoptosis detection system  according to the manufacturer’s protocol. HepG2 cells were seeded into 25 mL culture flasks at approximately 1 × 106 cells/mL and incubated for 24 h in a humidified atmosphere of 5% CO2 at 37 °C. Cells were treated with sulforaphene (in serum free media) at IC50 concentration of 72 h (33.8 μM). The cells were incubated for 24, 48 and 72 h in a humidified atmosphere of 5% CO2 at 37 °C. Treatment-free cells were grown as a negative control. After incubation, cells were harvested, added onto poly-l-lysine coated slides and air dried. The cells were then fixed with 4% paraformaldehyde solution in PBS for 25 min at room temperature. After fixing, the slides were washed with fresh PBS for 5 min and the cells were permeabilised by immersing the slides in 0.2% Triton® X-100 solution in PBS for 5 min. The cells were washed again with fresh PBS, mixed with 100 μL equilibration buffer and allowed to equilibrate at room temperature for 10 min. rTdT reaction mix (100 μL) was added to the equilibrated areas, covered with plastic cover slips and left to incubate at 37 °C for 60 min. To terminate the reactions, the slides were immersed in 2X SSC for 15 min at room temperature then washed with fresh PBS for 5 min. Slides were immersed in 0.3% hydrogen peroxide in PBS for 5 min to block endogenous peroxidases. The slides were again washed with PBS and mixed with 100 μL Streptavidin HRP solution per slide, and incubated for 30 min. Finally, 100 μL of DAB solution was added to each slide and allowed to stand until a light brown background appeared. Slides were subsequently viewed under a light microscope  to detect morphological changes.The terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay was carried out using the DeadEnd50 concentration of 72 h (33.8 μM). The cells were incubated for 24, 48 and 72 h in a humidified atmosphere of 5% CO2 at 37 °C. At the end of the incubation, cells were fixed with 4% paraformaldehyde and then permeabilised with Triton X-100 (0.1% in PBS). Cells were finally stained using DAPI in PBS (2.5 μg/mL) and allowed to stand for 20 min in a dark condition. Finally, morphological changes were viewed using fluorescence microscopy .4′,6-diamidino-2-phenylindole (DAPI) staining was carried out according to the method described by Papi et al.  with sli6 cells/ mLand incubated for 24 h in a humidified atmosphere of 5% CO2 at 37 °C. The cells were treated with sulforaphene (in serum free media) at IC50 concentration of 72 h (33.8 μM). The cells were incubated for 24, 48 and 72 h in a humidified atmosphere of 5% CO2 at 37 °C. Treatment-free cells were grown as a negative control. Cells were harvested with trypsin , centrifuged at 300× g for 10 min and kept cool on ice. Separately, 5 μL AO (10 mg/mL) was mixed with 5 μL PI (10 mg/mL) making a double-staining dye of AO/PI and kept in an ice bath in dark conditions. An aliquot of the cell suspensions (10 μL) was added to the dye mixture and transferred (5 μL) onto a glass slide for fluorescence viewing. Slides were observed under a UV-Fluorescence microscope within 30 min.The AO/PI morphological assessment of apoptotic cells was carried out according to the method of Arbab et al.  with min6 cells/ mL and incubated for 24 h. Cells were treated with sulforaphene (in serum free media) at an IC50 concentration of 72 h (33.8 μM). The cells were treated for 24, 48 and 72 h in a humidified atmosphere of 5% CO2 at 37 °C. Treatment-free cells were grown as negative controls. The cells were harvested with trypsin and centrifuged at 300× g for 10 min after treatment. The cells were then washed in PBS three times to remove any remaining media. Annexin V-FITC (5 μL) was added to 195 μL of cell suspension binding buffer (50 mL binding buffer and 150 mL distilled water), mixed and incubated in the dark for approximately 10 min. The cells were then washed and resuspended in 190 μL binding buffer (50 mL binding buffer and 150 mL distilled water) and finally 10 μL PI solution was added. The cells were kept on ice and then analysed by flow cytometry The Annexin V-FITC assay was carried out using the Annexin V-FITC assay kit (Sigma-Aldrich) according to the protocols provided. Briefly, HepG2 cells were seeded into 25 mL culture flasks at approximately 1 × 10TM 3/7, Caspase-GloTM 8 and Caspase-GloTM 9 assay kits . The assay was conducted following the manufacturer’s protocols. Briefly, HepG2 cells were cultured in a 96-well plate and incubated for 24 h in a humidified atmosphere of 5% CO2 at 37 °C. The cells were then treated with sulforaphene (in serum free media) at an IC50 concentration of 72 h (33.8 μM). Cells were then incubated for 24, 48 and 72 h. After treatment, 100 μL of assay reagent was added to each well and incubated for 1 h at room temperature. Finally, the luminescence was measured using a microplate reader .The activities of caspase-3/7, -8 and -9 were measured using a luminescence assay, with Caspase-GloTM PLUS DNA Reagent Kit  according to the protocols provided. HepG2 cells were seeded into 25 mL culture flasks at approximately 1 × 106 cells/ mL and incubated for 24 h in a humidified atmosphere of 5% CO2 at 37 °C. Cells were treated with sulforaphene (in serum free media) at IC50 concentration of 72 h (33.8 μM) for 24, 48 and 72 h. Treatment-free cells were grown as a negative control. The cells were harvested with trypsin and centrifuged at 300× g for 10 min after treatment. Subsequently 1 mL of buffer solution (provided in the kit) was added and resuspended by gentle vortexing. The cells were centrifuged at 300× g for 5 min and the supernatant was discarded. A further 1 mL buffer solution was added to resuspend the cells, and following centrifugation, the supernatant was discarded. This step was repeated for a third time and cells were immediately stained with propidium iodide (200 μL). After incubation for 10 min on ice, each sample was filtered through a 12 × 75 mm polypropylene tube and analysed on the flow cytometer .The cell cycle analysis was carried out using the CycleTESTp < 0.05 referred to statistical significance.Data are presented as means ± standard deviation. Statistical evaluation was performed by two-way ANOVA using Dunnett’s test. A 95% level of confidence was considered; thus, Toxicity of intact GRE and its hydrolysed product, sulforaphene, on HepG2 cells, was determined using the MTT assay method with cisplatin as the positive control. There was no apparent cytotoxic effect of GRE for at least up to 72 h of incubation and up to a concentration of 100 μM. Sulforaphene, however, suppressed cell growth .Initial MTT screening with sulforaphene for 24 h indicated significant growth inhibition in HepG2 cells compared to MCF-7 and HT-29 cells .In general, DAPI, TUNEL and AO/PI staining methods clearly showed induction of apoptosis in HepG2 cells after treatment with sulforaphene. HepG2 cells treated with sulforaphene showed clear apoptotic features which became more apparent as the treatment period increased . In the Aside from nuclear condensation, DAPI staining revealed that all treated HepG2 cells visibly appeared to lose cell structure with increasing time of incubation. The control cells, however, remained intact and evenly shaped. When treated for 24 h, most cells remained intact but lost their shape once the incubation period reached 48 h. Following treatment with sulforaphene for 72 h, all cells were completely ruptured. The DAPI staining noticeably showed apoptotic morphological changes in sulforaphene treated cells in terms of both nuclear condensation and cell structure loss.The AO/PI staining images displayed apoptotic changes in the sulforaphene treated cells in terms of colour and morphology. The apparent fluorescent colouration in the untreated cells compared to the treated cells was the first noticeable change. The control cells displayed a bright green colouration and an intact nuclear structure, indicating healthy viable cells, whereas treated cells exhibited a bright orange stain signifying the presence of apoptotic cells. With increasing incubation time with sulforaphene, the relative number of green cells gradually decreased while that of orange cells increased, indicating a shift from viable to apoptotic cells. At 72 h, the orange stain further turned to bright red indicating cell death and rupture.Similar to the DAPI staining, AO/PI staining also revealed a change in cell structure. After 24 h of incubation with sulforaphene, there were clear indications of chromatin condensation and blebbing of the HepG2 cell membrane. After a 48-h incubation, more chromatin condensation and blebbing were observed. Upon 72 h of incubation, small apoptotic bodies and dead cells were identified. The morphological changes as well as the shift from green to orange fluorescent cells that were observed from the AO/PI staining were sequential from 24 h up to 72 h of sulforaphene treatment, indicating gradual cell death via apoptosis with increasing length of exposure.Finally, the TUNEL assay also revealed clear apoptotic characteristics in sulforaphene-treated HepG2 cells. There was no visible colouration in untreated cells whereas in cells treated with sulforaphene, there were dark brown stains, which, similar to the other staining methods, increased in intensity with increasing duration of incubation. This change in staining denotes apparent DNA fragmentation and apoptotic nuclei while clear morphological changes in cell structure were also observed with increasing treatment period. In the period from 24 to 72 h, treated cells were observed to have gradually lost in cell integrity and eventually ruptured by the end of the incubation.The Annexin V assay revealed that sulforaphene induced early and late stage of apoptosis in HepG2 cells . The AnnIn the untreated cells, there was minimal cell distribution in Q1, Q2 and Q4 indicating a very low number of necrotic, early and late apoptotic cells, respectively. However, the cell distribution in these quadrants increased after treatment with sulforaphene. The percentage of early apoptotic cells (Q4) increased gradually with increasing treatment duration, being 1.67%, 10.58% and 13.81% following 24, 48 and 72 h of incubation, respectively. In the untreated cells, only 0.39% of cells were present in this quadrant. The untreated HepG2 cells showed a distribution of 0.60% for the late apoptotic cells (Q2). Similar to early apoptosis, the percentage of late apoptotic cells increased to 2.45%, 18.95% and 35.25% cells for 24, 48 and 72 h of sulforaphene treatment, respectively. The increase in cell distribution indicated a time dependent increase in late apoptotic cells, parallel to that noted in Q4.Lastly, necrotic cells, represented by Q1, displayed only a slight increase in cell distribution as a result of treatment with the isothiocyanate. In the untreated cells only a percentage of 0.57% necrotic cells was observed while in the sulforaphene-treated cells it increased to 0.77%, 3.51% and 2.66% for 24, 48 and 72 h incubation, respectively. Thus, the overall cell population shift indicated that sulforaphene caused a significant increase apoptosis in HepG2 cells.The treatment of HepG2 cells with sulforaphene for 24, 48 and 72 h significantly enhanced the activity of both caspase-3/7 and -9, but not caspase 8 .caspase-3/7, -8 and -9 enzyme activities were detected in HepG2. caspase-3/7 and -9 enzymes were elevated upon sulforaphene treatment, the extent of increase rising with the duration of the incubation. In contrast, caspase-8 enzymes were suppressed following the same treatment, with the effect being more pronounced as the incubation period increased.0/G1 phase but decreased to 56.05% after treatment with sulforaphene for 24 h; longer treatment further reduced the number of cells arrested in this phase; however, the HepG2 cell population was seen to be increasingly arrested at the SubG0/G1 phase with increasing sulforaphene treatment period. In the control cells, a cell population of only 2.65% was arrested in the SubG0/G1 phase. This population increased to 25.74%, 66.76% and 65.86% as the sulforaphene treatment duration was extended. The arrested cell population in the G2+M phase decreased from 8.42% in the untreated cells, to 2.83% for the cells treated up to 72 h, while there was no significant change in cell arrest in the S phase (from 4.31% in the control sample to 4.79% in the sample treated up to 72 h).The DNA content in sulforaphene-treated HepG2 cells was assessed using the cell cycle phase distribution analysis in a time dependent manner. The cell cycle arrest was analysed using flow cytometry, and Over the years, our understanding of apoptosis as well as the apoptotic controlling genes in relation to cancer therapy has increased dramatically ,22. In aIn particular, isothiocyanates, major derivatives of GLs, have been recognised as potential chemopreventive agents, acting through a number of mechanisms, including modulation of the various phases of cell growth . Sulfora50 values is relatively small among the three cell lines, there are other justifications for selecting HepG2 cells for further investigation. Firstly, in agreement with the present study, Sangthong et al. [The initial cytotoxicity screening showed that sulforaphene was most effective against HepG2 cells in comparison to HT29 and MCF-7 cells. Although the margin of ICg et al.  reportedRaphanus sativus and pure sulforaphene on liver cancer cells was performed, but their effect on apoptosis was not evaluated. Hence, based on these considerations, HepG2 was selected for further investigation.Thus, HepG2 cells may be sensitive to the structurally similar sulforaphene and sulforaphane. Previous studies focused on the potential of several other isothiocyanates, such as sulforaphane, benzyl isothiocyanate, phenylethyl isothiocyanate, and erucin, to inhibit proliferation and induce apoptosis in liver cancer cells ,31,33. F50 value of 33.8 μM when incubated with HepG2 cells for 72 h which is within the range of the value reported for most other isothiocyanates [50 values for several isothiocyanates following incubation with HepG2 cells differing in terms of concentration due to a dissimilar number of cells used and the incubation hours. This indicates that sulforaphene exhibits similar cytotoxicity in HepG2 cells to the extensively studied sulforaphane and many other isothiocyanates. The parent compound, GRE, did not display cytotoxicity in these cells even following a 72 h incubation in concordance with previous studies [Sulforaphene exhibited an ICcyanates ,33,34. T studies ,41,42,43An important attribute of chemopreventive agents is the ability to selectively induce the cell death of malignant cells via apoptosis and not through necrosis . IdentifIn validating further the apoptotic potential of sulforaphene, the annexin V-FITC assay was utilised which indicated a shift in cell distribution from viable to apoptotic after 72 h of treatment with sulforaphene, with a percentage shift of 13.81% and 35.25% cells for the early and late apoptotic stages, respectively, accompanied by a decline in cell proliferation. This cytotoxic and apoptotic potential of sulforaphene in HepG2 cells adds further to the findings by Sangthong et al. .The nature of the apoptotic mechanism leading to cell death is critical in determining a promising anticancer compound. Precise modulation of caspases is crucial to avoid unusual or premature apoptotic cell death which can potentially lead to carcinogenesis, autoimmunity, neurodegeneration, and immunodeficiency ,49. In t0/G1 peak in a cell population profile [0/G1 phase arrest in HepG2 cells, suggesting a reduced DNA content. The detection of subG0/G1 phase arrest occurs only if sufficient cellular DNA has been lost. Thus, sulforaphene has the potential to cause sufficient DNA loss to halt the cell cycle at the subG0/G1 phase in HepG2 cells.Another important attribute of a potential anticancer agent is the ability to initiate cell cycle arrest in cancer cells . The mol profile . Corresp0/G1 phase arrest in HepG2 cells alludes to a possible relationship with the intrinsic death pathway as indicated by the up-regulation of caspase-3/7 and -9 that may be linked to the generation of reactive oxygen species as similarly indicated by Papi et al. [The subGi et al. . Accordii et al.  the bioa0/G1 phase as compared to the G0/G1, G2 + M and S phases was noted.This article focuses on a study of the cytotoxicity and apoptotic potential of sulforaphene in HepG2 cells. Sulforaphene was cytotoxic in these cells whereas its precursor, the glucosinolate GRE, was inactive. Morphological analysis employing AOPI, DAPI and TUNEL staining assays revealed clear apoptotic activity. In further studies using Annexin V-FITC and caspase, analyses established significant increases in early and late apoptosis, and in the activity of caspases -3/7 and -9. Moreover, impairment of cell proliferation by cell cycle arrest at the Sub GThe present findings suggest positive apoptotic activity of sulforaphene in HepG2 cells via successive accompaniment of apoptosis subsequent to cell proliferation inhibition by the compound. These findings will contribute to the effort to identify phytochemicals that can potentially function as human anticancer agents.