A robust post-insertion method for the preparation of targeted siRNA LNPs.

Targeted delivery of nucleic acids is gaining momentum due to improved efficacy, selectivity, increased circulation time and enhanced tissue retention in target cells. Using nucleic acid-based therapies previously undruggable targets have proven now to be amenable for treatment. Currently, several methods for preparing targeted or labelled delivery vehicles for nucleic acids are based on liposomal formulations. Lipid nanoparticles (LNPs) are structurally different from liposomes and these methods should therefore be evaluated before being translated to siRNA LNPs preparation protocols. Here, we describe a robust and facile method for the preparation of targeted or fluorescently labelled siRNA LNPs. Using a copper free strain-promoted azide-alkyne cycloaddition (SPAAC) we demonstrate that post-insertion of ligand-lipid conjugates into preformed LNPs is superior to direct-surface modification because it preserves the physicochemical parameters of the LNPs. We found that the time point of solvent removal by dialysis is critical and affects the hydrodynamic diameter of the LNPs; post-insertion after dialysis shows the smallest increase in hydrodynamic diameter and polydispersity index (PDI). The post-insertion of ligand-lipid conjugates also proceeded with rapid kinetics and high efficacy over a wide temperature range. Using this optimised protocol, we generated siRNA LNPs containing both targeting and fluorescent tracking ligands allowing us to monitor siRNA LNP uptake kinetics in dependence of the targeting ligand. In aggregate, we describe a robust approach for the generation of targeted and labelled siRNA LNPs that allows their controlled and facile decoration with ligand combinations.

Double Heterotopic Pregnancies with Live Intrauterine Pregnancy after Ovarian Stimulation.

Heterotopic pregnancies, although rare in natural conceptions, have increased in incidence with use of assisted reproductive techniques (ART). Double heterotopic pregnancy in addition to an intrauterine pregnancy is exceedingly rare. In this case, we present a patient who underwent ovulation induction and intrauterine insemination (IUI) and was found on ultrasound to have a live heterotopic pregnancy. Intraoperatively, both fallopian tubes were grossly swollen and engorged. Bilateral salpingectomy was performed. Pathology identified gestational products in both fallopian tubes consistent with a double heterotopic pregnancy. Postoperatively, the intrauterine pregnancy resulted in a live birth. Although double heterotopic pregnancy and an intrauterine pregnancy is exceedingly rare, this case emphasizes the importance of routinely inspecting the contralateral fallopian tube at the time of surgery for an ectopic pregnancy, particularly in patients undergoing ovulation induction.

An ultra-rapid acoustic micromixer for synthesis of organic nanoparticles.

Mixing is a crucial step in many chemical analyses and synthesis processes, particularly in nanoparticle formation, where it determines the nucleation rate, homogeneity, and physicochemical characteristics of the products. In this study, we propose an energy-efficient acoustic platform based on boundary-driven acoustic streaming, which provides the rapid mixing required to control nanoprecipitation. The device encompasses oscillatory bubbles and sharp edges in the microchannel to transform the acoustic energy into vigorous vortical fluid motions. The combination of bubbles and sharp edges at their immediate proximity induced substantially stronger acoustic microstreams than the simple superposition of their effects. The device could effectively homogenize DI water and fluorescein within a mixing length of 25.2 µm up to a flow rate of 116 µL min-1 at a driving voltage of 40 Vpp, corresponding to a mixing time of 0.8 ms. This rapid mixing was employed to mitigate some complexities in nanoparticle synthesis, namely controlling nanoprecipitation and size, batch to batch variation, synthesis throughput, and clogging. Both polymeric nanoparticles and liposomes were synthesized in this platform and showed a smaller effective size and narrower size distribution in comparison to those obtained by a hydrodynamic flow focusing method. Through changing the mixing time, the effective size of the nanoparticles could be fine-tuned for both polymeric nanoparticles and liposomes. The rapid mixing and strong vortices prevent aggregation of nanoparticles, leading to a substantially higher throughput of liposomes in comparison with that by the hydrodynamic flow focusing method. The straightforward fabrication process of the system coupled with low power consumption, high-controllability, and rapid mixing time renders this mixer a practical platform for a myriad of nano and biotechnological applications.

Association between gastrointestinal cancers and fingerprint patterns in the Iranian population.

Gastrointestinal cancers are malignant diseases with high mortality rate. Early diagnosis of patients could improve the results of treatment. Many studies used dermatoglyphics as a biomarker to predict the incidence of genetic diseases and cancers. This study assessed the association between gastrointestinal cancers and particular fingerprint patterns, which could be useful in early diagnosis of these malignancies. The study was conducted on 153 histopathologically confirmed gastrointestinal cancer patients and 299 healthy individuals. The fingerprints were taken by a specific method of rolling the subject's fingers or thumbs in ink. The data were analyzed for the significance using the chi-square test and the t-test. Odds ratio with 95% confidence intervals were calculated. Dermatoglyphic analysis showed that whorl and loop patterns significantly changed in the case group as compared to control. However, the odds ratio suggested that whorl pattern in 6 or more fingers might be a risk factor for developing gastrointestinal cancers. Our results showed that there is an association between fingerprint patterns and gastrointestinal cancers, and so, the dermatoglyphic analysis may aid in the early diagnosis of these cancers.

The impact of motivational interviewing on participation in childbirth preparation classes and having a natural delivery: a randomised trial.

OBJECTIVE: This study aimed to determine the effectiveness of motivational interviewing on women's participation in childbirth classes and their subsequent natural vaginal delivery. DESIGN: Randomised controlled trial. SETTING: Prenatal clinic of the Shohada Women's Hospital, Behshahr, Mazandaran, Iran. POPULATION: This study was conducted with 230 nulliparous women. Participants were randomised into three groups, including 76 women in the motivational interviewing group, and 77 women in both the lecture and the control groups. METHODS: Participants were assessed at three time points, including at baseline (16-19 weeks of gestation) and then following the intervention (at 21 and 37 weeks of gestation). The motivational interviewing group received two focus interviews and two telephone follow-up sessions (at 3 and 6 weeks after the last session of motivational interviewing). The lecture group received a speech session. The control group received routine care service. MAIN OUTCOME MEASURES: Frequency of participation in childbirth preparation classes and mode of delivery. RESULTS: Over 90% of women in the motivational interviewing group participated in childbirth preparation classes, whereas the rate of participation in the lecture and the control groups was 59.7 and 27.3%, respectively. The probability of maternal participation in childbirth classes in the motivational interviewing and in the lecture groups was 3.3 (95% CI 2.1-4.5) and 2.2 (95% CI 1.4-3.0) times the probability of maternal participation in the control group, respectively. Moreover, the intervention groups had 1.4 (95% CI 1.1-1.8) and 1.1 (95% CI 0.9-1.4) times the probability of natural delivery, compared with the control group. The frequency of natural delivery in motivational interviewing, lecture, and control groups was 68.4, 54.5, and 48.1%, respectively. The results showed a statistically significant difference between the mean scores for the awareness and attitude scores between the three groups in different time periods. CONCLUSION: We found that motivational interviewing can be a useful tool for encouraging pregnant women to attend childbirth preparation classes. TWEETABLE ABSTRACT: Motivational interviewing with nulliparous women is strongly associated with their attendance in childbirth preparation classes.

Extraction of glycogen on mild condition lacks AIG fraction.

OBJECTIVE: Extraction of animal tissues with cold water or perchloric acid yields less glycogen than is obtained with hot-alkaline. Extraction with acid and alkaline gives two fractions, acid soluble (ASG) and insoluble glycogen (AIG). The aim of this work is to examine the hypothesis that not all liver glycogen is extractable by Tris-buffer using current techniques. MATERIALS AND METHODS: Rat liver was homogenized with Tris-buffer pH 8.3 and extracted for the glycogen fractions, ASG and AIG. The degree of homogenization was changed to remove all glycogen. RESULTS: The content of glycogen was 47.7 ± 1.2 and 11.6 ± 0.8 mg/g wet liver in the supernatant and pellet of the first extraction respectively. About 24% of total glycogen is lost through the first pellet. Increasing the extent of homogenization from 30 to 180 sec and from 15000 to 20000 rpm followed with 30 sec ultrasonication did not improve the extraction. ASG and AIG constitute about 77% and 23% of the pellet glycogen respectively. CONCLUSIONS: Extraction with cold Tris-buffer failed to extract glycogen completely.  Increasing the extent of homogenization followed with ultrasonication also did not improve the extraction. Thus it is necessary to re-examine the previous findings obtained by extraction with cold Tris-buffer.

Improvement of the classical assay method for liver glycogen fractions: ASG is the main and metabolic active fraction.

OBJECTIVE: Acid digestion of animal tissues yields two fractions of glycogen, acid soluble (ASG) and insoluble (AIG). The current study was performed to improve the assay method for glycogen fractions in rat liver in different physiological states. MATERIALS AND METHODS: All steps of the assay were manipulated and optimized to measure the content of ASG and AIG in fed and starved rat liver. RESULTS: In postmortem liver tissue, total glycogen was decreased slowly at 4°C and rapidly at 25°C but was well stabilized at -20°C and -70°C. At room temperature, ASG underwent autolysis at the rate of 1.3% and decreased by half at 35 min, while AIG increased slightly. The yield of the recovery of ASG during four successive extractions depends on the tissue concentration, and at the ratio of 50 mg tissue per 2 mL perchloric acid (PCA) was about 93.2%, 6.3%, 0.3% and 0.05% respectively. The increase in the time and extent of homogenization of the tissue with cold PCA and using ultrasonication had not any significant effect on the extraction yield of ASG. The time of centrifugation of the tissue extract could be reduced from 15 to 7.5 minutes with no significant decrease in the recovery of ASG. On extraction with ethanol, the yield of recovery of ASG reached the maximal level of 97.5% at a final ethanol concentration of 60%. The recovery of ASG was not improved in the presence of KCl. During 24 starvation, total glycogen depleted completely and the change occurred entirely in ASG, while AIG did not change significantly. CONCLUSIONS: The CV% was less than 5% for the optimized assays of glycogen fractions. ASG is the main and metabolically active portion of glycogen in rat liver.

Homogenization versus homogenization-free method to measure muscle glycogen fractions.

OBJECTIVE: The glycogen is extracted from animal tissues with or without homogenization using cold perchloric acid. Three methods were compared for determination of glycogen in rat muscle at different physiological states. MATERIALS AND METHODS: Two groups of five rats were kept at rest or 45 minutes muscular activity. The glycogen fractions were extracted and measured by using three methods. RESULTS: The data of homogenization method shows that total glycogen decreased following 45 min physical activity and the change occurred entirely in acid soluble glycogen (ASG), while AIG did not change significantly. Similar results were obtained by using "total-glycogen-fractionation methods". The findings of "homogenization-free method" indicate that the acid insoluble fraction (AIG) was the main portion of muscle glycogen and the majority of changes occurred in AIG fraction. CONCLUSIONS: The results of "homogenization method" are identical with "total glycogen fractionation", but differ with "homogenization-free" protocol. The ASG fraction is the major portion of muscle glycogen and is more metabolically active form.

A new protocol for separation of acid soluble and insoluble fractions from total glycogen and simultaneous measurements.

OBJECTIVE: The glycogen is extracted routinely from animal tissues with cold perchloric acid (PCA). Acid soluble glycogen (ASG) is extracted, while the insoluble fraction (AIG) is liberated using hot alkaline. The current study was performed to separate and measure ASG, AIG and total glycogen in the same sample simultaneously. MATERIALS AND METHODS: The protocol has the four phases of tissue digestion, extraction, separation of fractions and measurement. The liver tissue was weighed and digested with four volumes of 30% KOH and heated in boiling water bath for 10 min. Total glycogen was extracted with ethanol at a final concentration of 55%. The suspension of total glycogen was separated into the two fractions of acid soluble and insoluble by adding of 30 µL PCA (70%) followed by a short and mild centrifugation. Total glycogen, ASG and AIG have derived from the same sample and analyzed for glucose. RESULTS: Analysis of different weights of the liver tissue using the current procedure shows that the fractions of glycogen are measured accurately. The CV% was less than 5% for inter- and intra-assays of total glycogen and ASG. The CV% was more than 5% for inter-assays of AIG, but it lessened in intra-assays. During 24 h starvation, total glycogen depleted completely (71.4 ± 8.3 mg/g wet vs. 4.4 ± 1.2, p ≤ 0.004) and the change occurred entirely in ASG (66.9 ± 7.8 vs. 1.9 ± 1.1, p ≤ 0.004), while AIG did not change significantly (4.4 ± 1.3 vs. 2.2 ± 0.9, p ≤ 0.08). CONCLUSIONS: The values of ASG, AIG and total glycogen obtained by the current protocol are the same as the classical homogenization method but the procedure is more easy and precise. ASG is the main and metabolically active portion of glycogen in rat liver.

Immune response in susceptible BALB/c mice immunized with DNA encoding Lipophosphoglycan 3 of Leishmania infantum.

Visceral leishmaniasis is a serious parasitic infection that the development of an effective vaccine is necessary to control the disease. Lipophosphoglycan 3 (LPG3) is essential for the synthesis of glycoconjugates as parasite virulence factors. In this study, we evaluated the immunogenicity of Leishmania infantum LPG3 gene as a DNA vaccine against murine visceral leishmaniasis. For this purpose, BALB/c mice were immunized subcutaneously with the DNA encoding LPG3 either alone or in combination with recombinant heat shock protein 70 (rHSP70). Next, its immunogenicity and protective efficacy were evaluated in the immunized mice. The results showed a mixed Th1/Th2 response following immunization, which was associated with the production of both IFN-γ and IL-10 by splenocytes compared with control groups but did not lead to reduction in the splenic parasite burden. Serum levels of IgG antibody isotypes indicated no significant difference between the LPG3 DNA and the empty vector. In addition, the co-administration of rHSP70 with the DNA vaccine offered no additive protective advantage on experimental infectious challenge. Thus, we propose to strengthen the immunogenic potential of L. infantum LPG3 in prime-boost approach with a powerful adjuvant to elicit a robust parasite-specific protective Th1 response.

Characterization and improvement of phenol-sulfuric acid microassay for glucose-based glycogen.

OBJECTIVES: Phenol-sulfuric acid reagent is used to measure the concentration of glyco-polymers and -conjugates. There are several uncertainties on glycogen measurement in the tissues. We aimed to improve phenol-sulfuric reagent for microassay of glucose based-glycogen in small tube or microplate. MATERIALS AND METHODS: The condition of the reaction was optimized and scaled down for both small tube and microplate application. RESULTS: The color intensity was found to be a function of all components of the assay mixture, that is, the amount of sugar and phenol together with the volume of total water and acid. The absorbance increased in the range of 4-10 mg of phenol and reached the plateau between 10-16 mg per 1 mL of acid. The color intensity was a linear function of total water volume (sugar-water- phenol). The sensitivity increased eight times as total water volume was changed from 50 up to 400 µL. The curve for acid volume peaked at about 1 mL. The optimal assay condition was determined to be 13 mg of phenol (200 µL 6.5%), 400-425 µL of total water volume (100 µL of sugar, 100 µL water) for 1 mL of acid. The initial spontaneous high temperature is essential the reaction to proceed and any handling gives inconsistent results and decreases the precision and sensitivity of the method. The values were scaled down by a factor of 0.5 for tube application and reading in cuvet or microplate and by 0.2 or 0.15 for microplate application. CONCLUSIONS: The results indicated that phenol-sulfuric acid reagent could be scaled down to 1.0, 0.5 and 0.20, 0.15 mL of sulfuric acid for microassay of glucose based-glycogen.

Determining dimensions of iranians' individual social health: a qualitative approach.

BACKGROUND: Social health is important to be assessed as a dimension of health. The aim of study was to determine domains and sub-domains of individual social health of Iranians. METHODS: This study was carried out with a qualitative approach, using thematic content analysis. Twenty five experts participated in interviews, using individual semi-structured interviews between November 2010 and June 2011.This data supported with strong search. RESULTS: Two main areas extracted from these interviews including social support and social function. The social support domain contained seventeen sub-areas, including social support at the time of disease; disability; daily life issues, etc. And the social function as second domain contained twelve sub-areas, including: financial aids to others; emotionally aids to others; participating in social groups, etc. CONCLUSION: We developed a conceptual framework for social health in the individual level in Iranian population. It makes preparations for providing a valid and reliable measurement scale for social health in next studies and evidence-based policy-making.

Simultaneous-bilateral TKA: double trouble - affirms.

In this paper, we will consider the current role of simultaneous-bilateral TKA. Based on available evidence, it is our opinion that simultaneous bilateral TKA carries a higher risk of morbidity and mortality and should be reserved for select few.

Association of interleukin-15 single nucleotide polymorphisms with resistance to brucellosis among Iranian patients.

Interleukin (IL)-15, a Th1-related cytokine, triggers inflammatory cells' recruitment and increases the expression of interferon gamma (IFN-γ), which is an important cytokine in the immunity against brucellosis. Different single-nucleotide polymorphisms (SNPs) have been observed in the IL-15 gene, so this study aimed to investigate the probable association between these SNPs and susceptibility to brucellosis among Iranian patients. A total of 190 patients with brucellosis and 83 healthy milk farmers who consumed contaminated raw milk and dairy products from animals involved with brucellosis were included in this study. All the patients and the controls were genotyped for four IL-15 polymorphisms at positions 267, 367, 13687 and 14035 using polymerase chain reaction-restriction fragment length polymorphisms. The 267C and 13687A alleles, haplotypes CGCT and CAAA and the 267CC and 13687AA genotypes were significantly more frequent in the controls than in the patients (P = 0.014, 0.03, 0.006, 0.024, 0.026 and 0.01, respectively), so the variation in the IL-15 gene may be one of the factors affecting the resistance to brucellosis. In contrast, the frequency of haplotypes CGCA and TACT was significantly higher in patients compared with controls (P = 0.015 and 0.007, respectively), and interestingly the last one was observed only in the patients; therefore, it may serve as a predictive factor for brucellosis. In conclusion, it could be suggested that IL-15 genetic variants can affect resistance or susceptibility to human brucellosis among Iranian patients.

Epidemiology of injuries and poisonings in emergency departments in Iran.

OBJECTIVES: There are few reports on epidemiological patterns of injury and injury-related mortality in developing countries. This study aimed to report the epidemiology of injuries and poisonings in emergency departments in Iran. STUDY DESIGN: Retrospective study using available data from 20 March 2005 to 19 March 2008. METHODS: Recorded Injury Surveillance System (ISS) data including demographics, place of residence, type of injury, and outcome during emergency department stay were extracted from the databank of the national ISS and included in the final analysis. RESULTS: In total, 2,991,624 emergency department admissions due to injury were recorded at university hospitals during the study period. According to the national census in 2006, Iran had a population of 70,472,846, so the injury admission rate to university hospital emergency departments was 1.4%/year in Iran. The mean age of the patients was 26.5 [standard deviation (SD) 16.9] years, and 72.7% of the cases were male. The most common cause of injury was road traffic accidents (RTAs) (31.9%), followed by hit (25.5%) and falls (10.9%). Intoxication was associated with 5.3% of all injuries. The overall emergency department mortality rate was 0.6%. Of those who died, the mean age was 32.6 (SD 21.1) years. All fatal injuries, except burn injuries, were more common in males. Intoxication-related deaths occurred in 3.8% of cases. In patients aged <13, 13-65 and >65 years, hit (28.2%), RTAs (34%) and RTAs (27.9%) were, respectively, the most common causes of injury. In all age groups, RTAs were the most common cause of death. CONCLUSIONS: This study determined the epidemiology of injuries and poisonings in emergency departments in Iran. The mortality rate in this study was low in comparison with other research, which may be explained in the context of inappropriate prehospital or interhospital care in Iran. This finding can be employed to formulate targeted preventive strategies based on the incidence of the more common types of injury.

Synbiotic preparation in men suffering from functional constipation: a randomised controlled trial.

BACKGROUND: Evaluating the effects of a commercially available synbiotic preparation (contains both prebiotic and probiotic elements) on functional constipation in males. METHODS: In a randomised controlled trial, a total of 66 adult men with functional constipation were equally allocated to receive a synbiotic mixture or a placebo. The synbiotic mixture or placebo was given as capsules with the same shape and colour, and patients received the capsules twice a day for 4 weeks. RESULTS: A total of 60 patients (31 in the synbiotic group) completed the study. At baseline evaluation, there was no significant difference between the mean stool frequency per week in synbiotic and placebo groups [mean difference of 0.11 times (95% CI: -0.31-0.55), p = 0.58]. However, mean stool frequency increased significantly at weeks 2 [mean difference of 1.32 times (95% CI: 0.21-2.43)] and 4 [mean difference of 1.58 times (95% CI: 0.18-2.99)] in the synbiotic group compared with the placebo group (p = 0.02). A significant difference (p = 0.006) was found at weeks 2 [mean difference of 0.83 (95% CI: 0.20-1.45)] and 4 [mean difference of 0.91 (95% CI: 0.3-1.51)] between the synbiotic and placebo groups regarding the Bristol stool form score. No adverse effect was seen in the synbiotic group. CONCLUSION: The results of this study indicated that this specific commercial product seemed to be effective in increasing stool frequency and improving consistency in this sample of males with functional constipation. However, further studies with longer follow ups, and including females and elderly patients are required to confirm the efficacy of this product for treatment of functional constipation.

Role of dipstick in detection of haeme pigment due to rhabdomyolysis in victims of Bam earthquake.

Avoiding life-threatening complications of rhabdomyolysis depends on early diagnosis and prompt management. The aim of this study was to evaluate the role of urinary dipstick test in the detection of haeme pigment in patients who were at risk of acute renal failure (ARF) due to rhabdomyolysis after suffering injury in the Bam earthquake. Serum creatine phosphokinase (CPK) level was used as the gold standard for prediction of ARF. ARF developed in 8 (10%) of 79 patients studied. We found no significant differences in the sensitivity, specificity and accuracy of dipstick urine and serum CPK tests for identifying patients who were at risk of ARF. However, dipstick urine test is an easy test that can be performed quickly at an earthquake site.

Burden of spinal cord injury in Tehran, Iran.

STUDY DESIGN: Investigation of burden of traumatic spinal cord injury (SCI) using disease modeling. OBJECTIVES: The present paper is intended to estimate the SCI burden for the year 2008. SETTING: Tehran, capital of Iran. METHODS: Epidemiological data needed to calculate Disability-Adjusted Life-Years (DALYs) for SCI, was estimated according to prevalence, duration and relative risk of mortality using DISMOD software. For DALY calculation, the years of life lost because of premature mortality (YLL) was added to the number of years lost because of disability (YLD). To calculate DALYs for SCI, first year DALY calculated separately and for the next years, the DALY was assessed for six different clinical presentations of traumatic SCI including quadriplegia, quadriparesis, paraplegia, paraparesis, hemiplegia and hemiparesis. RESULTS: In first year following SCI, the DALY was 3772 years, which has 0.5 DALY per 1000 people and YLL/DALY was 89.3%. Following the first year, the DALY was 435 for quadriplegia, 163 for quadriparesis, 868 for paraplegia, 164 for paraparesis, 26 for hemiplegia and 14 for hemiparesis. The total YLL for traumatic SCI was 4077 years and total YLD was 1364 years (total YLL/DALY was 74.9%) and total DALY was 5441 years, (M/F=2.0), which has 0.7 DALY per 1000 people in Tehran in 2008. CONCLUSIONS: This study showed a high burden for SCI. Identifying the risk factors of SCI, and performing cost-effective preventive interventions for reducing burden of SCI is recommended.

Role of dipstick in detection of haeme pigment due to rhabdomyolysis in victims of Bam earthquake

Avoiding life-threatening complications of rhabdomyolysis depends on early diagnosis and prompt management. The aim of this study was to evaluate the role of urinary dipstick test in the detection of haeme pigment in patients who were at risk of acute renal failure [ARF] due to rhabdomyolysis after suffering injury in the Bam earthquake. Serum creatine phosphokinase [CPK] level was used as the gold standard for prediction of ARF. ARF developed in 8 [10%] of 79 patients studied. We found no significant differences in the sensitivity, specificity and accuracy of dipstick urine and serum CPK tests for identifying patients who were at risk of ARF. However, dipstick urine test is an easy test that can be performed quickly at an earthquake site