Separation of U87 glioblastoma cell-derived small and medium extracellular vesicles using elasto-inertial flow focusing (a spiral channel).

Nanoscale and microscale cell-derived extracellular vesicle types and subtypes are of significant interest to researchers in biology and medicine. Extracellular vesicles (EVs) have diagnostic and therapeutic potential in terms of biomarker and nanomedicine applications. To enable such applications, EVs must be isolated from biological fluids or separated from other EV types. Developing methods to fractionate EVs is of great importance to EV researchers. Our goal was to begin to develop a device that would separate medium EVs (mEVs, traditionally termed microvesicles or shedding vesicles) and small EVs (sEVs, traditionally termed exosomes) by elasto-inertial effect. We sought to develop a miniaturized technology that works similar to and provides the benefits of differential ultracentrifugation but is more suitable for EV-based microfluidic applications. The aim of this study was to determine whether we could use elasto-inertial focusing to re-isolate and recover U87 mEVs and sEVs from a mixture of mEVs and sEVs isolated initially by one round of differential ultracentrifugation. The studied spiral channel device can continuously process 5 ml of sample fluid per hour. Using the channel, sEVs and mEVs were recovered and re-isolated from a mixture of U87 glioma cell-derived mEVs and sEVs pre-isolated by one round of differential ultracentrifugation. Following two passes through the spiral channel, approximately 55% of sEVs were recovered with 6% contamination by mEVs (the recovered sEVs contained 6% of the total mEVs). In contrast, recovery of U87 mEVs and sEVs re-isolated using a typical second centrifugation wash step was only 8% and 53%, respectively. The spiral channel also performed similar to differential ultracentrifugation in reisolating sEVs while significantly improving mEV reisolation from a mixture of U87 sEVs and mEVs. Ultimately this technology can also be coupled to other microfluidic EV isolation methods in series and/or parallel to improve isolation and minimize loss of EV subtypes.

Effects of second responder programs on repeat incidents of family abuse: An updated systematic review and meta-analysis.

Background: Family abuse is a recurrent phenomenon within a select population of households. This form of abuse can include any physical or psychological harassment that occurs between family or household members, and often involves complex mental and emotional issues that are resistant to intervention. Traditional criminal justice strategies for combating this issue have evolved over time but have frequently demonstrated limited success. Within the past few decades, multiagency programs to address repeat family abuse have gained popularity. One such program, termed "second responders," teams police officers with social service workers, victim advocates, or counselors to conduct follow-up visits with victims of family abuse following a complaint. Second responders seek to educate victims about the cyclical nature of family abuse, engage in safety planning, and/or provide service referrals. These interventions are based on the premise that victims are more likely to be receptive to crime prevention opportunities immediately following victimization. Second responder interventions have received support from the US Department of Justice and their adoption has spread in both the United States and internationally, however, there remains little conclusive evidence on their effects. Objectives: To update and extend the findings of the prior second responders systematic review and meta-analysis by synthesizing the results of published and unpublished second responder evaluations through October of 2021. This review also examines the use of victim services as a secondary outcome and incorporates a number of additional moderator analyses. Search Methods: The Global Policing Database (GPD), a repository of all experimental and quasi-experimental evaluations of policing interventions conducted since 1950, was searched using keywords related to second responder interventions and repeat family violence from 2004 to December 2019 (https://gpd.uq.edu.au/s/gpd/page/about). This search was also supplemented with additional strategies, such as reference harvesting of prior reviews, searching 2020 and 2021 volumes of leading academic journals, reviewing the reference lists of eligible studies, searching additional gray literature repositories focused on domestic violence, and consulting with eligible study authors. Selection Criteria: Eligible studies were required to include a treatment group that received the second responder intervention and a comparison group that did not. Assignment to these conditions could be either experimental or quasi-experimental, but quasi-experimental studies were required to use either matched comparison groups or multivariate analysis methods to control for confounding factors. Eligibility was limited to studies reporting on at least one measure of repeat family abuse, such as intimate partner violence, elder abuse, or general family abuse. Measures of repeat abuse could be based on either official (i.e., police data) or unofficial (i.e., victim survey data) data sources. Data Collection and Analysis: Five new studies were identified between 2004 and 2019, all of which contained sufficient data for the calculation of at least one effect size. Along with the 10 studies included in the prior review, a total of 15 studies and 29 distinct effect sizes were analyzed across three outcome constructs. Effect sizes were calculated as logged odds ratios and results were synthesized using random effects models with restricted maximum likelihood estimation. Final results were exponentiated to represent the percentage point difference in the odds of a given outcome for treatment groups relative to control groups. Risk of bias was assessed using items adapted from the Cochrane Risk of Bias tools for experimental and quasi-experimental studies. Eligible studies were generally considered to be of low risk of bias, however, issues with survey success/contact rates and the analytical approaches to these problems led to concern in several studies. Results: These analyses suggest that second responder interventions produced no significant effects on either police or victim-reported measures of repeat family abuse, in aggregate. However, findings from the more rigorous experimental studies indicated that second responder interventions were associated with a statistically significant 22% (95% confidence interval [CI] [1.04, 1.43]) increase in the odds of a police-reported repeat family abuse incident, with no significant variability in individual study results. Additionally, studies that measured the use of victim services as a secondary outcome were associated with a statistically significant 9% (95% CI [1.02, 1.16]) increase in the odds of service use for treatment groups relative to control groups. Several study characteristics also proved to be important moderators of treatment effects. Increases in the speed of the second response were associated with significant decreases in the odds of a victim-reported repeat incident, and studies that measured repeat family abuse using households were associated with significantly higher odds of a police-reported repeat incident, compared to studies that used the same victim or victim/offender pairing more generally. Authors' Conclusions: Second responder interventions are undoubtedly appealing based on their logic and intentions. Yet, well-intentioned programs with sound logic can still backfire, and the results of this updated review provide evidence that may be suggestive of a backfire effect. Even so, any firm conclusions from this review are limited by a lack of knowledge on the mechanisms operating in between the implementation of the second response intervention and the observed effects, as well as the small sample sizes involved in many analyses. While it seems clear that these programs are not producing any broad reductions in self-reported victimization, the increase in police-reported violence seen in experimental studies could indicate either a true increase in abuse or an increased willingness to call the police. The lack of observed impact on victim-reported violence would suggest the latter, but without more specific measures, such conclusions should be avoided. If these results are indicative of increased reporting, however, many may consider this a desirable outcome, particularly given the often-underreported nature of family abuse and the potential for increased reporting to lead to long-term reductions in abuse. Furthermore, these results provide an indication that second responder programs can produce other intended effects, such as increasing the retention of victim services, and that the specific characteristics of these interventions may moderate their effects. It is unclear why elements such as the immediacy of the second response or the unit of analysis being evaluated would impact study results, but these observations are consistent with the theory that domestic violence interventions must capitalize on short windows of opportunity and create separation between victims and offenders to reduce exposure and subsequent victimization. This potential indicates a need for more research on second responder programs, but specifically research that examines these moderating characteristics and mechanisms. Even in light of this potential, second responder programs do not, on average, appear to reduce the prevalence of repeat family abuse. Given the presence of alternative (and possibly more effective) domestic violence interventions that now exist (e.g., Safe Dates, Shifting Boundaries, Green Dot, etc.), it seems that policymakers may wish to look elsewhere for efforts to reduce family abuse.

Development and Testing of a Continuous Flow-Electrical-Split-Flow Lateral Transport Thin Separation System (Fl-El-SPLITT).

In this work, a new high-volume, continuous particle separation device that separates based upon size and charge is described. Two continuous flow-electrical-split-flow lateral transport thin (Fl-El-SPLITT) device architectures (a platinum electrode on a porous membrane and a porous graphite electrode under a membrane) were developed and shown to improve particle separations over a purely electrical-SPLITT device. The graphite FL-El-SPLITT device architecture achieved the best separation of approximately 60% of small (28 nm) vs large (1000 nm) polystyrene particles. Fl-El-SPLITT (platinum) achieved a 75% separation on a single pass using these same particles. Fl-El-SPLITT (platinum) achieved a moderate 26% continuous separation of U87 glioma cell-derived small extracellular vesicles (EVs) from medium EVs. Control parameter testing showed that El-SPLITT continuously directed particle motility within a channel to exit a selected port based upon the applied voltage using either direct current or alternating current. The transition from one port to the other was dependent upon the voltage applied. Both large and small polystyrene particles transitioned together rather than separating at each of the applied voltages. These data present the first ever validation of El-SPLITT in continuous versus batch format. The Fl-El-SPLITT device architecture, monitoring, and electrical and fluid interfacing systems are described in detail for the first time. Capabilities afforded to the system by the flow addition include enhanced particle separation as well as the ability to filter out small particles or desalinate fluids. High-throughput continuous separations based upon electrophoretic mobility will be streamlined by this new technique that combines electrical and flow fields into a single device.

An automated instrument for intrauterine insemination sperm preparation.

Sperm preparation is critical to achieving a successful intrauterine insemination and requires the processing of a semen sample to remove white blood cells, wash away seminal plasma, and reduce sample volume. We present an automated instrument capable of performing a sperm preparation starting with a diluted semen sample. We compare our device against a density gradient centrifugation by processing 0.5 mL portions of patient samples through each treatment. In 5 min of operating time, the instrument recovers an average of 86% of all sperm and 82% of progressively motile sperm from the original sample while removing white blood cells, replacing the seminal plasma, and reducing the volume of the sample to the clinically required level. In 25 min of operating time, density gradient centrifugation recovers an average of 33% of all sperm and 41% of progressively motile sperm. The automated instrument could improve access to IUI as a treatment option by allowing satellite doctor's offices to offer intrauterine insemination as an option for patients without the clinical support required by existing methods.

Characterization of Human Glioblastoma versus Normal Plasma-Derived Extracellular Vesicles Preisolated by Differential Centrifugation Using Cyclical Electrical Field-Flow Fractionation.

Although many properties for small extracellular vesicles (sEVs, formerly termed "exosomes") isolated at ∼100 000g are known, a wide range of values are reported for their electrophoretic mobility (EM) measurements. This paper reports for the first time the effect of dilution on the EM of U87 glioblastoma cell-derived and plasma-derived sEVs and medium size EVs (mEVs, commonly termed "oncosomes") preisolated by differential centrifugation. Furthermore, the effect of resalting on the EM of sEVs and mEVs was evaluated. The EM of U87 sEVs and U87 mEVs showed an increase as the salt concentration decreased to 0.005% of the initial salt concentration. However, for the plasma sEVs and plasma mEVs, the electrophoretic mobility increased as the salt concentration decreased to 0.01% of the initial salt concentration and then increased to its initial value when the salt concentration decreased to 0.005% of the initial salt concentration. For both U87 and plasma sEVs and mEVs, the EM remained almost constant when the concentration of the particles changed and the salt concentration was kept the same as its initial value. This indicates that the EM of EVs is only a function of the salt concentration of the buffer and is independent of the concentration of the particles. The sEVs and mEVs were separated with cyclical ElFFF for the first time. The results indicate that ElFFF was able to fractionate the EVs, and a crescent-shaped trend was found for the retention time when the applied AC voltage was altered (increased).

Exosome Isolation: Cyclical Electrical Field Flow Fractionation in Low-Ionic-Strength Fluids.

The influence of buffer substitution and dilution effects on exosome size and electrophoretic mobility were shown for the first time. Cyclical electrical field flow fractionation (Cy-El-FFF) in various substituted fluids was applied to exosomes and other particles. Tested carrier fluids of deionized (DI) water, 1× phosphate buffered saline (PBS), 0.308 M trehalose, and 2% isopropyl alcohol (IPA) influenced Cy-El-FFF-mediated isolation of A375 melanoma exosomes. All fractograms revealed a crescent-shaped trend in retention times with increasing voltage with the maximum retention time at ∼1.3 V AC. A375 melanoma exosome recovery was approximately 70-80% after each buffer substitution, and recovery was independent of whether the sample was substituted into 1× PBS or DI water. Exosome dilution in deionized water produced a U-shaped dependence on electrophoretic mobility. The effect of dilution using 1× PBS buffer revealed a very gradual change in electrophoretic mobility of exosomes from ∼-1.6 to -0.1 µm cm/s V, as exosome concentration was decreased. This differed from the use of DI water, where a large change from ∼-5.5 to -0.1 µm cm/s V over the same dilution range was observed. Fractograms of separated A375 melanoma exosomes in two substituted low-ionic-strength buffers were compared with synthetic particle fractograms. Overall, the ability of Cy-El-FFF to separate exosomes based on their size and charge is a highly promising, label-free approach to initially catalogue and purify exosome subtypes for biobanking as well as to enable further exosome subtype interrogations.

A Pathology of Mesh and Time: Dysejaculation, Sexual Pain, and Orchialgia Resulting From Polypropylene Mesh Erosion Into the Spermatic Cord.

OBJECTIVE: The ubiquitous use of polypropylene mesh in hernia surgery has spawned a new clinical syndrome: chronic post-herniorrhaphy neuralgia. A subset of that clinical picture is dysejaculation, sexual pain, and orchialgia. We propose to identify the processes that lead to that pain. SUMMARY OF BACKGROUND DATA: Specimens of vas adherent to polypropylene mesh, explanted in an attempt to control severe, life-changing inguinodynia are extremely difficult to obtain. This scarcity may be due to ingrained attitudes in our society about removal of vas and/or testicles for whatever reason. Attempts at preserving such damaged structures may paradoxically contribute to the chronicity and severity of such pain. METHODS: The medical files of patients who had mesh specimens explanted because of severe chronic post-herniorrhaphy pain were reviewed to identify cases with recorded evidence, at the time of surgery, of involvement of spermatic cord/vas deferens with mesh. These criteria were met in 13 cases and the specimens were analyzed histologically. RESULTS: The vas deferens was resected in 83% (5 of 6) of the patients with a history of sexual pain and/or dysejaculation (vs 14% of those without a history of sexual pain, P = 0.03). Histology demonstrated unequivocal mesh invasion of the spermatic cord, where the initial damage occurred to nerves (autonomic, somatic), then to the smooth muscle of the vas while the lumen remained patent. In 50% (3 of 6), the vas and other cord structures appeared to be completely invaded by the mesh and replaced by scar tissue. CONCLUSIONS: Irreversible damage of the nerves and vas musculature due to mesh migration is one of the mechanisms for sexual pain and dysejaculation. Attempts at all cost to preserve elements of the spermatic cord may not be justified in cases of severe pain, especially sexual pain (and/or dysejaculation) and intraoperative finding of cord involvement by the mesh. Vasectomy with mesh removal may well be indicated and be considered not a radical procedure but a conservative measure given the severity of the pain!

A review of exosome separation techniques and characterization of B16-F10 mouse melanoma exosomes with AF4-UV-MALS-DLS-TEM.

Exosomes participate in cancer metastasis, but studying them presents unique challenges as a result of their small size and purification difficulties. Asymmetrical field flow fractionation with in-line ultraviolet absorbance, dynamic light scattering, and multi-angle light scattering was applied to the size separation and characterization of non-labeled B16-F10 exosomes from an aggressive mouse melanoma cell culture line. Fractions were collected and further analyzed using batch mode dynamic light scattering, transmission electron microscopy and compared with known size standards. Fractogram peak positions and computed radii show good agreement between samples and across fractions. Ultraviolet absorbance fractograms in combination with transmission electron micrographs were able to resolve subtle heterogeneity of vesicle retention times between separate batches of B16-F10 exosomes collected several weeks apart. Further, asymmetrical field flow fractionation also effectively separated B16-F10 exosomes into vesicle subpopulations by size. Overall, the flow field flow fractionation instrument combined with multiple detectors was able to rapidly characterize and separate exosomes to a degree not previously demonstrated. These approaches have the potential to facilitate a greater understanding of exosome function by subtype, as well as ultimately allow for "label-free" isolation of large scale clinical exosomes for the purpose of developing future exosome-based diagnostics and therapeutics.