Marker-specific sorting of rare cells using dielectrophoresis

Current techniques in high-speed cell sorting are limited by the inherent coupling among three competing parameters of performance: throughput, purity, and rare cell recovery. Microfluidics provides an alternate strategy to decouple these parameters through the use of arrayed devices that operate in parallel. To efficiently isolate rare cells from complex mixtures, an electrokinetic sorting methodology was developed that exploits dielectrophoresis (DEP) in microfluidic channels. In this approach, the dielectrophoretic amplitude response of rare target cells is modulated by labeling cells with particles that differ in polarization response. Cell mixtures were interrogated in the DEP-activated cell sorter in a continuous-flow manner, wherein the electric fields were engineered to achieve efficient separation between the dielectrophoretically labeled and unlabeled cells. To demonstrate the efficiency of marker-specific cell separation, DEP-activated cell sorting (DACS) was applied for affinity-based enrichment of rare bacteria expressing a specific surface marker from an excess of nontarget bacteria that do not express this marker. Rare target cells were enriched by >200-fold in a single round of sorting at a single-channel throughput of 10,000 cells per second. DACS offers the potential for automated, surface marker-specific cell sorting in a disposable format that is capable of simultaneously achieving high throughput, purity, and rare cell recovery.     

Caregiver- and Child-Reported Anxiety Using an Autism-Specific Measure: Measurement Properties and Correlates of the Anxiety Scale for Children with Autism Spectrum Disorder (ASC-ASD) in Verbal Young People with ASD

Identifying and measuring anxiety in young people on the autism spectrum can be challenging. The present study investigated the use of the Anxiety Scale for Children with Autism Spectrum Disorder (ASC-ASD), a self- and caregiver-rated screening tool in a Singaporean sample of ninety-one verbal autistic youths and their caregivers. Internal consistency ranged from satisfactory to desirable (α?=?.74–.92). Convergent validity with medium-large effect size was established using a structured diagnostic interview, the Mini-International Neuropsychiatric Interview for Children and Adolescents (MINI-KID). ASC-ASD scores were positively associated with autistic symptoms and response patterns indicated strong endorsement of autism-specific items. The findings are discussed in relation to existing literature on assessment of anxiety in ASD and in light of the study’s strengths and limitations.

     

Circumferential Resection Margin Involvement in Stage III Rectal Cancer Patients Treated with Curative Resection Followed by Chemoradiotherapy: A Surrogate Marker for Local Recurrence?

Purpose

Circumferential resection margin (CRM) involvement is a well-known predictor for poor prognosis in rectal cancer. However, the significance is controversial in some studies. Accordingly, this study attempted to examine the prognostic impact of CRM involvement in stage III rectal cancer.

Materials and Methods

Between January 1990 and December 2007, a total of 449 patients who underwent curative resection followed by complete adjuvant chemoradiotherapy for stage III rectal cancer located within 12 cm from the anal verge were selected. Patients were divided into a CRM-positive group (n=79, 17.6%) and a CRM-negative group (n=370, 82.4%).

Results

With a median follow-up of 56.6 months, recurrent disease was seen in 53.2 and 43.5% of the CRM-positive and CRM-negative group, respectively. CRM involvement was an independent prognostic factor for 5-year systemic recurrence-free survival (HR: 1.5, CI: 1.0-2.2, p=0.017). However, no significant difference was observed for local recurrence rate between the two groups (13.0 and 13.5%, respectively, p=0.677).

Conclusion

In this study, local recurrence rate did not differ according to CRM involvement status in stage III rectal cancer patients, although CRM involvement was shown to be an independent poor prognostic factor. Accordingly, validation of the results of this study by further large prospective randomized trials is warranted.     

Infection with Toxoplasma gondii does not elicit predator aversion in male mice nor increase their attractiveness in terms of mate choice

Behavioral manipulation hypothesis posits that some parasites induce behavioral changes in the host to increase transmission efficiency of the parasite. Protozoan parasite Toxoplasma gondii infecting rats has been widely studied in this context. T. gondii increases attractiveness of infected male rats and reduces innate aversion of rats to cat odor, likely increasing transmission of the parasite by sexual and trophic routes respectively. It is currently unexplored if T. gondii induces gain of male attractiveness in experimental models other than rats. Here we show that laboratory infection of two strains of mice does not induce behavioral manipulation. Moreover, T. gondii infection results in reduction of male attractiveness in one of the strains. In agreement with this observation, T. gondii infection also fails to induce reduction in innate aversion to cat odors in mice. Effects of the parasite on mice mate choice are similar to effects of several other parasites in this animal model. Thus, behavioral change induced by the parasite may be specific to the rodent species.     

Isoflavone Genistein Induces Fluid Secretion and Morphological Changes in the Uteri of Post-Pubertal Rats

A reported increase in the incidence of infertility following high genistein intake could be related to alteration in the normal fluid volume and morphology of the uterus in adult female. In view of this, we investigated the effect of this compound on fluid secretion, fluid volume and morphology of the uterus in post-pubertal rats. Methods: Ovariectomised SD rats were treated with 17-β oestradiol (E) (0.8 X 10^-4 mg/kg/day) and genistein (0.5, 5, 10, 25, 50 and 100 mg/kg/day) for three days. Following drug treatment, in-vivo uterine perfusion was performed and the rate of fluid secretion and the volume of fluid in the uterus were determined via changes in weight (μl/min) and F-dextran concentration of the perfusate respectively. The animals were then sacrificed and the uteri were removed for weight determination, morphological analyses and proliferative cell nuclear antigen (PCNA) expression analyses by Western blotting. Results: Subcutaneous genistein treatment resulted in a dose-dependent increase in fluid secretion rate, fluid volume and uterine wet weight. Treatment with 100 mg/kg/day genistein resulted in a remarkable increase in the rate of uterine fluid secretion, the volume of the uterine luminal fluid as well as the circumference of the uterine and uterine glandular lumen suggesting an excessive fluid accumulation. Meanwhile, there were evidence of glandular hyperplasia and an increase in the expression of PCNA following treatment with 50 and 100 mg/kg/day genistein. Conclusion: High genistein intake could potentially cause adverse effects on the uterus by inducing excessive fluid secretion and accumulation as well as hyperplasia.     

Bisphosphonate-associated peri-implant fractures: a new clinical entity?: A series of 10 patients with 11 fractures

Background and purpose

The current definition of atypical femoral fractures (AFFs) associated with bisphosphonate use includes only de novo fractures. However, in recent years reports of bisphosphonate-associated periprosthetic fractures involving stemmed arthroplasty implants have emerged. In a case series of peri-implant fractures in femurs with plate/screw constructs, we aimed to assess similarities with classical AFFs and how their location may have implications for the pathogenesis and management of AFFs.

Patients and methods

We retrospectively identified 10 patients with 11 peri-implant fractures.

Results

The patients were ambulant women, mean age 80 (70–92) years. Mean duration of bisphosphonate use was 5 (1–10) years. The peri-implant fractures were sustained an average of 4 years (6 months to 9 years) from the time of index surgery. They were all associated with low-energy mechanisms. 8 fractures occurred near the tip of a plate, while 3 traversed the penultimate screwhole of a plate. The peri-implant fractures showed clinical and radiological features of atypicality such as lateral cortical thickening, simple fracture pattern, and lack of comminution. The patients underwent revision surgery, with bone grafting used in all but 1 case. Radiological union was evident after 2–4 months.

Interpretation

Atypical peri-implant fractures of the femur associated with bisphosphonate use may be a new entity. Stress lesions and atypical fractures may tend to develop over stress risers along the operated femur. This finding has implications for the pathogenesis and clinical management of AFFs.Bisphosphonates form the cornerstone of antiresorptive therapy in the management of post-menopausal osteoporosis. They are used in the treatment of malignant and osteoclast-mediated metabolic bone disease. Their use in patients who have undergone total joint arthroplasty of the lower limb is associated with higher periprosthetic bone mineral density and longer implant survival (Bhandari et al. 2005). Bisphosphonates exert their therapeutic effect by reducing bone turnover and increasing overall mineralization. This translates to increased bone mineral density and bone strength, corresponding clinically to reduced risk of vertebral and non-vertebral fragility fractures (Black et al. 1996).In recent years, several published reports have described atypical femoral fractures (AFFs) of the proximal femoral diaphysis and subtrochanteric region, in association with bisphosphonate use (Goh et al. 2007, Neviaser et al. 2008, Isaacs et al. 2010). Bisphosphonates are associated with a higher age-adjusted relative risk of AFF in women than in men, which is higher in alendronate users than in risedronate users (Schilcher et al. 2015). Bisphosphonates may cause changes in bone matrix composition and bone mechanical properties, increasing the propensity for accumulation of microdamage. Impaired target remodeling would contribute to the progression of macrocracks. High interfragmentary strain from physiological loads at a thin fracture line may be a mechanical factor in lack of bone healing (Aspenberg et al. 2010).Periprosthetic/peri-implant fractures are currently excluded from the definition of AFFs. We suggest that peri-implant fractures of the femur with features of atypicality may be linked to bisphosphonate use and that they should be recognized as a clinical entity.     

Growth Factor Measurement and Histological Analysis in Platelet Rich Fibrin: A Pilot Study

Objective

The aim of this study was to compare growth factor amount contained in platelet rich fibrin (PRF) and compare with that in platelet rich plasma (PRP), and in whole blood. And also to investigate distribution of growth factors and cellular components in PRF.

Materials and Methods

PRF and PRP were obtained from the same sample of peripheral blood. Extraction of proteins were done with lysis buffer, accompanied by freeze and thaw procedures. Concentration of two representative growth factors in platelets: platelet derived growth factor (PDGF) and transforming growth factor beta (TGF-β), were measured with enzyme-linked immunosorbent assay (ELISA). PRF was cut into three parts: (top, middle and bottom), and growth factor concentration was measured respectively. Paraffin embedded section of PRF was observed with Giemsa stain. Immuno-histochemical analysis with anti-PDGF and anti-TGF-β antibodies was also conducted.

Results

The growth factor levels in PRF was higher than in peripheral blood and comparable to those in PRP. Growth factor levels in bottom part of PRF was much higher than in top and middle part. Microscopically, platelets and mono-nucleated cells were concentrated just above the yellow–red interface. Poly-nucleated cells were concentrated below the interface.

Conclusion

The growth factors were surely concentrated in PRF. This result can support basis of good clinical outcomes. For effective application of PRF, the knowledge that growth factors and cells are not equally distributed in PRF should be utilized.     

Trends and risk factors of elderly-onset Crohn's disease: A nationwide cohort study

BACKGROUND The incidence of inflammatory bowel disease(IBD)is increasing in Asia.Numerous risk factors associated with IBD development have been investigated.AIM To investigate trends and environmental risk factors of Crohn’s disease(CD)diagnosed in persons aged≥40 years in South Korea.METHODS Using the National Health Insurance Service database,a total of 14060821 persons aged>40 years who underwent national health screening in 2009 were followed up until December 2017.Patients with newly diagnosed CD were enrolled and compared with non-CD cohort.CD was identified according to the International Classification of Diseases 10th revision and the rare/intractable disease registration program codes from the National Health Insurance Service database.The mean follow-up periods was 7.39 years.Age,sex,diabetes,hypertension,smoking,alcohol consumption,regular exercise,body mass index,anemia,chronic kidney disease(CKD)and dyslipidemia were adjusted for in the multivariate analysis model.RESULTS During the follow-up,1337(1.33/100000)patients developed CD.Men in the middle-aged group(40-64 years)had a higher risk than women[adjusted hazard ratio(aHR)1.46,95%confidence interval(CI):1.29-1.66];however,this difference tended to disappear as the age of onset increases.In the middle-aged group,patients with a history of smoking[(aHR 1.46,95%CI:1.19-1.79)and anemia(aHR 1.85,95%CI:1.55-2.20)]had a significantly higher CD risk.In the elderly group(age,≥65 years),ex-smoking and anemia also increased the CD risk(aHR 1.68,95%CI:1.22-2.30)and 1.84(95%CI:1.47-2.30,respectively).Especially in the middle-aged group,those with CKD had a statistically elevated CD risk(aHR 1.37,95%CI:1.05-1.79).Alcohol consumption and higher body mass index showed negative association trend with CD incidence in both of the age groups.[Middle-aged:aHR 0.77(95%CI:0.66-0.89)and aHR 0.73(95% CI:0.63-0.84),respectively][Elderly-group:aHR 0.57(95% CI:0.42-0.78)and aHR 0.84(95%CI 0.67-1.04),respectively].For regular physical activity and dyslipidemia,negative correlation between CD incidences was proved only in the middle-aged group[aHR 0.88(95%CI:0.77-0.89)and aHR 0.81(95%CI:0.68-0.96),respectively].CONCLUSION History of cigarette smoking,anemia,underweight and CKD are possible risk factors for CD in Asians aged>40 years.