Sheath fluid impacts the depletion of cellular metabolites in cells afflicted by sorting induced cellular stress (SICS).

Flow cytometrists have long observed a spectrum of cell-type-specific changes ranging from minor functional defects to outright cell destruction after purification of cells using conventional droplet cell sorters. We have described this spectrum of cell perturbations as sorter induced cellular stress, or SICS (Lopez and Hulspas, Cytometry, 2020, 97, 105-106). Despite the potential impact of this issue and ubiquitous anecdotes, little has been reported about this phenomenon in the literature, and the underlying mechanism has been elusive. Inspired by others' observations (Llufrio et al., Redox Biology, 2018, 16, 381-387 and Binek et al., Journal of Proteome Research, 2019, 18, 169-181), we set out to examine SICS at the metabolic level and use this information to propose a working model. Using representative suspension (Jurkat) and adherent (NIH/3T3) cell lines we observed broad and consistent metabolic perturbations after sorting using a high-speed droplet cell sorter. Our results suggest that the SICS metabolic phenotype is a common cell-type-independent manifestation and may be the harbinger of a wide-range of functional defects either directly related to metabolism, or cell stress response pathways. We further demonstrate a proof of concept that a modification to the fluidic environment (complete media used as sheath fluid) in a droplet cell sorter can largely rescue the intracellular markers of SICS, and that this rescue is not due to a contribution of metabolites found in media. Future studies will focus on characterizing the potential electro-physical mechanisms inherent to the droplet cell sorting process to determine the major contributors to the SICS mechanism.

MiSet RFC Standards: Defining a Universal Minimum Set of Standards Required for Reproducibility and Rigor in Research Flow Cytometry Experiments.

Poor adherence to best practices, insufficient training, and pressure to produce data quickly may lead to publications of suboptimal biomedical research flow cytometry data, which contributes to the body of irreproducible research findings. In addition, documentation of compliance with best flow cytometry practices for submission, visualization, and publication of flow cytometry data is currently endorsed by very few scientific journals, which is particularly concerning as numerous peer-reviewed flow cytometry publications emphasize instrumentation, experimental design, and data analysis as important sources of variability. Guidelines and resources for adequate reporting, annotation and deposition of flow cytometry experiments are provided by MIFlowCyt and the FlowRepository database, and comprehensive expert recommendations covering principles and techniques of field-specific flow cytometry applications have been published. To facilitate the integration of quality-defining parameters into manuscript and grant submission and publication requirements across biomedical fields that rely on the use of flow-cytometry-based techniques, a single comprehensive yet easily and universally applicable document is needed. To produce such a list of gold-standard parameters that assess whether a research flow cytometry experiment has been planned, conducted, interpreted, and reported at the highest standard, a new initiative defining the minimum set of standards a robust and rigorous research flow experiment must fulfill (MiSet RFC Standards) was proposed at CYTO 2019. MiSet RFC Standards will integrate and simplify existing resources to provide a universal benchmark a flow cytometry experiment can easily be measured against. The goal of MiSET RFC Standards is its integration into peer-review and publication procedures through partnership with stakeholders, journals and publishers in biomedical and translational research. This article introduces the aims and anticipated timeline and discusses strategies for interdisciplinary consensus and implementation. A single-resource broadly applicable guideline will harmonize standards across different fields of biomedical research and lead to publication of more robust research findings. © 2019 International Society for Advancement of Cytometry.

Pro-tumorigenic effects of miR-31 loss in mesothelioma.

The human genome encodes several hundred microRNA (miRNA) genes that produce small (21-23n) single strand regulatory RNA molecules. Although abnormal expression of miRNAs has been linked to cancer progression, the mechanisms of this dysregulation are poorly understood. Malignant mesothelioma (MM) of pleura is an aggressive and highly lethal cancer resistant to conventional therapies. We and others previously linked loss of the 9p21.3 chromosome in MM with short time to tumor recurrence. In this study, we report that MM cell lines derived from patients with more aggressive disease fail to express miR-31, a microRNA recently linked with suppression of breast cancer metastases. We further demonstrate that this loss is due to homozygous deletion of the miR-31-encoding gene that resides in 9p21.3. Functional assessment of miR-31 activity revealed its ability to inhibit proliferation, migration, invasion, and clonogenicity of MM cells. Re-introduction of miR-31 suppressed the cell cycle and inhibited expression of multiple factors involved in cooperative maintenance of DNA replication and cell cycle progression, including pro-survival phosphatase PPP6C, which was previously associated with chemotherapy and radiation therapy resistance, and maintenance of chromosomal stability. PPP6C, whose mRNA is distinguished with three miR-31-binding sites in its 3'-untranslated region, was consistently down-regulated by miR-31 introduction and up-regulated in clinical MM specimens as compared with matched normal tissues. Taken together, our data suggest that tumor-suppressive propensity of miR-31 can be used for development of new therapies against mesothelioma and other cancers that show loss of the 9p21.3 chromosome.

Dynamics of Antibacterial Drone Establishment in Staphylococcus aureus: Unexpected Effects of Antibiotic Resistance Genes.

The antibacterial drone (ABD) system is based on repurposing the phage-inducible staphylococcal pathogenicity islands (SaPIs) for use as antibacterial agents that are indifferent to antibiotic resistance. The ABDs were constructed by inserting tetM for tetracycline resistance (Tcr) selection, replacing the SaPI virulence genes with bactericidal or bacteriostatic genes such as CRISPR/cas9/agrA, whose expression kills by double-strand cleavage of agrA, or CRISPR/dcas9/agrP2P3, whose expression blocks the target organism's virulence. ABD DNA is packaged in phage-like particles that attack their staphylococcal targets in vivo as well as in vitro. We determine ABD titers by transfer frequency, enumerate surviving cells as a function of multiplicity, and analyze the fate of ABD DNA with green fluorescent protein. An initial study revealed surprisingly that many more cells were killed by the ABD than were measured by transduction. Our study of this phenomenon has revealed several important features of the ABD system: (i) a significant number of entering ABD DNA molecules do not go on to establish stable transductants (i.e., are abortive); (ii) ABD cargo genes are expressed immediately following entry, even by the abortive ABDs; (iii) immediate plating on Tc-containing agar seriously underestimates particle numbers, partly owing to Tc inhibition of protein synthesis; (iv) replacement of tetM with cadA (conferring resistance to CdCl2) provides more accurate particle enumeration; (v) ABDs expressing CRISPR/cas9/agrA kill ∼99.99% of infected cells and provide the most accurate measurement of particle numbers as well as proof of principle for the system; and (vi) surprisingly, TetM interferes with stable establishment of ABD DNA independently of Tcr. IMPORTANCE For a particulate therapeutic agent, such as the ABD, accurate enumeration of particles is critical to enable evaluation of preparative procedures and calculation of therapeutic dosages. It is equally important that a selective marker used for these two purposes be biologically inert. We have long used tetM for these purposes but show here that tetM not only underestimates particle titers, by over 20-fold in some experiments, but also seriously impedes stable establishment of the therapeutic particle DNA. Given that tetM is a very convenient and widely used selective marker, publication of these findings is of considerable importance to the microbiological community as well as an interesting illustration of the unpredictable biological effects of genes taken out of their native context.

Diversity of Functionally Distinct Clonal Sets of Human Conventional Memory B Cells That Bind Staphylococcal Protein A.

Staphylococcus aureus, a common cause of serious and often fatal infections, is well-armed with secreted factors that disarm host immune defenses. Highly expressed in vivo during infection, Staphylococcal protein A (SpA) is reported to also contribute to nasal colonization that can be a prelude to invasive infection. Co-evolution with the host immune system has provided SpA with an Fc-antibody binding site, and a Fab-binding site responsible for non-immune superantigen interactions via germline-encoded surfaces expressed on many human BCRs. We wondered whether the recurrent exposures to S. aureus commonly experienced by adults, result in the accumulation of memory B-cell responses to other determinants on SpA. We therefore isolated SpA-specific class-switched memory B cells, and characterized their encoding VH : VL antibody genes. In SpA-reactive memory B cells, we confirmed a striking bias in usage for VH genes, which retain the surface that mediates the SpA-superantigen interaction. We postulate these interactions reflect co-evolution of the host immune system and SpA, which during infection results in immune recruitment of an extraordinarily high prevalence of B cells in the repertoire that subverts the augmentation of protective defenses. Herein, we provide the first evidence that human memory responses are supplemented by B-cell clones, and circulating-antibodies, that bind to SpA determinants independent of the non-immune Fc- and Fab-binding sites. In parallel, we demonstrate that healthy individuals, and patients recovering from S. aureus infection, both have circulating antibodies with these conventional binding specificities. These findings rationalize the potential utility of incorporating specially engineered SpA proteins into a protective vaccine.

Diaphragmatic injuries: what has changed over a 20-year period?

Traumatic diaphragmatic injuries are uncommon events but are associated with a high mortality. We hypothesize that injury pattern has changed over time with increasing prevalence of blunt injuries. A retrospective chart review was performed of 124 patients who sustained traumatic diaphragmatic injuries over the 20-year period between January 1, 1986 and December 31, 2005. Penetrating trauma accounted for 65 per cent (80/124) of all diaphragm injuries, and blunt trauma for 35 per cent (44/124). Mean Injury Severity Scores of 19 +/- 9 and 34 +/- 13 were observed for the penetrating and blunt trauma groups, respectively (P = 0.001). Blunt traumatic diaphragm injuries increased from 13 per cent in the first 10-year period to 66 per cent in the second 10-year period (P = 0.001). The overall mortality was 9 per cent (11/124) with 10 deaths resulting from blunt trauma and one resulting from penetrating trauma (P < 0.001). The mortality rate increased from 3 to 17 per cent over the two decades (P = 0.007). Our data suggests that over the last 20 years, the increase in mortality associated with traumatic diaphragmatic injury is primarily related to an increase in the proportion of patients with blunt trauma as a cause of their diaphragmatic injury and associated injuries.

Evaluating the Effects of Cell Sorting on Gene Expression.

Cell sorting is a commonly used technology to isolate highly purified cell populations for downstream applications. Because the sorted cells are destined for further analysis, i.e., gene expression assays or functional assays, ensuring that the sorting process itself has little effect on the cells is of utmost importance. Previous studies examining the effects of sorting on cellular function have primarily focused on a specific cell type or condition. One of the goals of the Flow Cytometry Research Group of the Association of Biomolecular Resource Facilities is to establish best practice guidelines for cell sorting conditions that minimize cell stress, perturbation, or injury to the sorted cell population. In this study, the effects of nozzle size, sample pressure, UV exposure, and instrument type were evaluated for their effects on gene expression and cell cycle using both established cell lines and primary cells across several flow cytometry shared facilities. Results indicate that nozzle size and pressure, as well as UV exposure and instrument type, have only minor effects on gene expression, which were diminished by subsequent culturing of the sorted cells. In this assessment, these data demonstrate that cell sorting itself, regardless of instrumentation used, has minimal effects on downstream cellular applications.

Evaluating the Effects of Cell Sorting on Gene Expression.

Cell sorting is a commonly used technology to isolate highly purified cell populations for downstream applications. Because the sorted cells are destined for further analysis, i.e., gene expression assays or functional assays, ensuring that the sorting process itself has little effect on the cells is of utmost importance. Previous studies examining the effects of sorting on cellular function have primarily focused on a specific cell type or condition. One of the goals of the Flow Cytometry Research Group of the Association of Biomolecular Resource Facilities is to establish best practice guidelines for cell sorting conditions that minimize cell stress, perturbation, or injury to the sorted cell population. In this study, the effects of nozzle size, sample pressure, UV exposure, and instrument type were evaluated for their effects on gene expression and cell cycle using both established cell lines and primary cells across several flow cytometry shared facilities. Results indicate that nozzle size and pressure, as well as UV exposure and instrument type, have only minor effects on gene expression, which were diminished by subsequent culturing of the sorted cells. In this assessment, these data demonstrate that cell sorting itself, regardless of instrumentation used, has minimal effects on downstream cellular applications.

Development of a sleeve gastrectomy weight loss model in obese Zucker rats.

BACKGROUND: Obesity promotes the development of diabetes and cardiovascular disease. The most effective weight loss treatment is bariatric surgery, but results greatly vary depending on the procedure. Sleeve gastrectomy (SG) has recently emerged as a reduced risk weight loss procedure for super obese patients. However, the mechanism of weight loss from SG and its effects on obesity-induced complications are yet to be determined. Our goal was to develop an experimental model of SG in genetically obese rats. MATERIALS AND METHODS: Male obese Zucker rats (400-500 g, leptin-insensitive) were anesthetized with isoflurane. After a midline laparotomy, the stomach was clamped, the greater curvature was excised, and a triple suture line was used to close the gastric remnant. Sham rats underwent laparotomy only. Metabolic parameters were followed for 14 d after surgery. RESULTS: Caloric intake and body weight decreased in SG rats over 14 d by 98 +/- 10 kcal/d and 74 +/- 14 g, respectively. Blood total cholesterol levels were lower in rats that lost weight. Furthermore, blood glucose levels were lower in rats that lost weight. Active ghrelin levels were unchanged in SG rats 14 d after surgery. CONCLUSIONS: These results show that SG promotes weight loss in obese Zucker rats. Furthermore, SG-induced weight loss is accompanied by improved plasma cholesterol and glucose profile. However, SG does not promote a prolonged decrease in ghrelin levels. These results suggest that SG is an effective weight loss procedure in leptin insensitivity to improve the lipid profile and decrease insulin resistance and these effects might be independent of changes in ghrelin levels.

Computed tomography grading systems poorly predict the need for intervention after spleen and liver injuries.

Computed tomography (CT) grading systems are often used clinically to forecast the need for interventions after abdominal trauma with solid organ injuries. We compared spleen and liver CT grading methods to determine their utility in predicting the need for operative intervention or angiographic embolization. Abdominal CT scans of 300 patients with spleen injuries, liver injuries, or both were evaluated by five trauma faculty members blinded to clinical outcomes. Studies were graded by American Association for the Surgery of Trauma criteria, a novel splenic injury CT grading system, and a novel liver injury grading system. The sensitivity and specificity of each methodology in predicting the need for intervention were calculated. The kappa statistic was used to determine interrater variability. Twenty-one per cent (39/189) of patients with splenic injuries visible on CT scans required interventions, whereas 14 per cent (21/154) of patients with liver injuries visible on CT required interventions. The overall sensitivity of all grading systems in predicting the need for surgery or angioembolization of the spleen or liver was poor; the specificity seemed to be fairly good. When evaluators were compared, the strength of agreement for the various scoring systems was only moderate. Anatomic CT grading systems are ineffective screening tools for excluding the need for operation or embolization after splenic or hepatic trauma. Although insensitive, CT is a good predictor (highly specific) of the need for intervention if certain definitive abnormalities are identified. Considerable inconsistency exists in interpretation of abdominal CT scans after trauma, even among experienced clinicians.

Survey on Scientific Shared Resource Rigor and Reproducibility.

Shared scientific resources, also known as core facilities, support a significant portion of the research conducted at biomolecular research institutions. The Association of Biomolecular Resource Facilities (ABRF) established the Committee on Core Rigor and Reproducibility (CCoRRe) to further its mission of integrating advanced technologies, education, and communication in the operations of shared scientific resources in support of reproducible research. In order to first assess the needs of the scientific shared resource community, the CCoRRe solicited feedback from ABRF members via a survey. The purpose of the survey was to gain information on how U.S. National Institutes of Health (NIH) initiatives on advancing scientific rigor and reproducibility influenced current services and new technology development. In addition, the survey aimed to identify the challenges and opportunities related to implementation of new reporting requirements and to identify new practices and resources needed to ensure rigorous research. The results revealed a surprising unfamiliarity with the NIH guidelines. Many of the perceived challenges to the effective implementation of best practices (i.e., those designed to ensure rigor and reproducibility) were similarly noted as a challenge to effective provision of support services in a core setting. Further, most cores routinely use best practices and offer services that support rigor and reproducibility. These services include access to well-maintained instrumentation and training on experimental design and data analysis as well as data management. Feedback from this survey will enable the ABRF to build better educational resources and share critical best-practice guidelines. These resources will become important tools to the core community and the researchers they serve to impact rigor and transparency across the range of science and technology.

Inspiratory Capacity and Vital Capacity of Healthy Subjects 9-81 Years of Age at Moderate-High Altitude.

BACKGROUND: Measurements of inspiratory capacity (IC) and vital capacity (VC) are used to recognize dynamic hyperinflation, but appropriate reference values are required to achieve accurate clinical interpretations. Altitude above sea level is a potential determining factor for lung volumes, including IC and VC. OBJECTIVE: To describe IC and VC for healthy people who live in Mexico City at an altitude of 2,240 m above sea level. METHODS: Healthy subjects ages 9-81 y completed slow spirometry by following 2005 American Thoracic Society/European Respiratory Society standards. Once associations were explored, linear regression models were constructed and values were compared with those from previously published equations. RESULTS: A total of 441 healthy subjects (55.1% women) participated. The mean age was 32 y (minimum age, 9 y; maximum age, 81 y). IC and VC measurements were associated with sex, age, height, and weight. An accelerated increase in IC and VC was evident from 9 to 20 y of age, followed by a gradual decrease in both sexes. In general, IC was higher in our population than predicted by previously published reference equations. CONCLUSIONS: IC in healthy people at 2,240 m above sea level was higher than that of previous reports about European and Latin-American subjects of the same height, sex, and age who were at sea level. The present study provided robust reference values for persons who lived at a moderate altitude.

Prevalence of sleep apnea in morbidly obese patients who presented for weight loss surgery evaluation: more evidence for routine screening for obstructive sleep apnea before weight loss surgery.

The incidence of obstructive sleep apnea has been underestimated in morbidly obese patients who present for evaluation for weight loss surgery. This retrospective study shows that the incidence of obstructive sleep apnea in this patient population is greater than 70 per cent and increases in incidence as the body mass index increases. Obstructive sleep apnea (OSA) is a common comorbidity in obese patients who present for evaluation for gastric bypass surgery. The incidence of sleep apnea in obese patients has been reported to be as high as 40 per cent. A retrospective review of our prospectively collected database was performed. All patients being evaluated for weight loss surgery for obesity were screened preoperatively for OSA using a sleep study. The overall incidence of sleep apnea in our patients was 78 per cent (227 of 290). All 227 were diagnosed by formal sleep study. There were 63 (22%) males and 227 (78%) females. The mean age was 43 years (range, 17-75 years). The mean body mass index (BMI) was 52 kg/m2 (range, 31-94 kg/m2). The prevalence of OSA in the severely obese group (BMI 35-39.9 kg/m2) was 71 per cent. For the morbidly obese group (BMI 40-40.9 kg/m2), the prevalence was 74 per cent and for the superobese group (BMI 50-59.9 kg/m2) 77 per cent. Those with a BMI 60 kg/m2 or greater, the prevalence of OSA rose to 95 per cent. The incidence of sleep apnea in patients presenting for weight loss surgery was greater than 70 per cent in our study. Patients presenting for weight loss surgery should undergo a formal sleep study to diagnose OSA before bariatric surgery.

How can trauma surgeons maintain their operative skills?

BACKGROUND: The operative experience of the dedicated trauma surgeon is declining. Much attention has focused on the operative workload of trauma surgeons as it is critical in both maintaining operative skills and promoting the interest of surgical residents in trauma careers. We examined the operative experience of our surgical service which includes trauma, emergency general surgery, and elective general surgery to analyze changes occurring over the past decade. METHODS: A retrospective study was performed by extracting data from the operative database at our Level I trauma center from January 1995 to December 2005. The cases were classified as trauma, emergency general surgery, or elective general surgery. Data were analyzed using weighted linear regression to analyze statistical significance. RESULTS: Although the total number of cases performed by the trauma service remained constant, the proportion of initial operative trauma cases (<24 hours from arrival to operation) decreased from 14% to 8% (r2 = 0.91, p < 0.001) over the study period. In contrast, emergency general surgery cases increased over this time period (r2 = 0.57, p < 0.01). Elective case volume was unchanged. The majority of the waning of trauma cases was due to decreased surgery on the liver and spleen and fewer neck explorations. CONCLUSIONS: Trauma operative experience decreased but emergency general surgery increased over a decade at our trauma center. It appears possible to maintain a busy operative trauma service by the inclusion of emergency general surgery consultations.

Risk Factors for Dysphagia Following a Cervical Fusion in a Trauma Population.

Background Dysphagia following a cervical fusion is a known complication; however, this has not been examined in the trauma population. We sought to identify risk factors that can be optimized in this population. Methods We performed a retrospective chart review on consecutive trauma patients who underwent a cervical fusion from 2014 to 2017 at a single institution with multiple surgeons. We included patients more than 18-years-old who were admitted through the emergency department with a diagnosis of acute cervical injury and underwent a cervical fusion during the same admission. We excluded patients who remained intubated postoperatively or underwent a tracheostomy. The primary outcome was dysphagia as evaluated by a bedside swallow test on postoperative day one by the nursing staff. This was followed by a standardized assessment performed by a speech therapist on postoperative day two in some cases. Variables of interest included sex, age, mechanism of injury, surgical approach, cervical levels, and Charlson comorbidity index. Univariate analysis was also utilized. Results Sixty patients met the study criteria. Nineteen patients (31.7%) developed dysphagia postoperatively. Mechanical falls were the most common injury mechanism (80%) and most surgical procedures were performed on the subaxial cervical spine (68.3%). Comparing the dysphagia groups, there was no significant difference among the confounding variables. Patients with dysphagia had an increased length of stay (10.6 ± 6.7 vs. 7.4 ± 3.1, p = 0.056) and were more likely to have had an anterior vs. posterior cervical fusion (63.2% vs. 34.1%, p = 0.056). Conclusions We found no statistically significant risk factors leading to postoperative dysphagia. The objective of this pilot is to find the baseline dysphagia rate and the potential modifiable factors in this unique patient population undergoing cervical fusion procedures.

Nascent Induced Pluripotent Stem Cells Efficiently Generate Entirely iPSC-Derived Mice while Expressing Differentiation-Associated Genes.

The ability of induced pluripotent stem cells (iPSCs) to differentiate into all adult cell types makes them attractive for research and regenerative medicine; however, it remains unknown when and how this capacity is established. We characterized the acquisition of developmental pluripotency in a suitable reprogramming system to show that iPSCs prior to passaging become capable of generating all tissues upon injection into preimplantation embryos. The developmental potential of nascent iPSCs is comparable to or even surpasses that of established pluripotent cells. Further functional assays and genome-wide molecular analyses suggest that cells acquiring developmental pluripotency exhibit a unique combination of properties that distinguish them from canonical naive and primed pluripotency states. These include reduced clonal self-renewal potential and the elevated expression of differentiation-associated transcriptional regulators. Our observations close a gap in the understanding of induced pluripotency and provide an improved roadmap of cellular reprogramming with ramifications for the use of iPSCs.

Outpatient complications encountered following Roux-en-Y gastric bypass.

Practitioners taking care of postoperative bariatric patients need to keep in mind all of the complications that this population faces to prevent unnecessary morbidity. Bariatric patients presenting postoperatively with abdominal pain, tachycardia, vomiting, tachypnea, and a sense of impending doom should be worked up aggressively to find the cause of their symptoms. Because the incidence of obesity is rising in children and adults, more patients will have surgery to help with their weight loss. Physicians caring for these patients must be able to diagnosis and treat their complications quickly and efficiently to prevent further complications.