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Introduction: Manual gating of flow cytometry (FCM) data for marrow cell analysis is a standard approach in current practice, although it is time- and labor-consuming. Recent advances in cytometry technology have led to significant efforts in developing partially or fully automated analysis methods. Although multiple supervised and unsupervised FCM data analysis algorithms have been developed, they have not been widely adopted by the clinical and research laboratories. In this study, we evaluated flowDensity, an open source freely available algorithm. as an automated analysis tool for classification of lymphocyte subsets in the bone marrow biopsy specimens. Materials and Methods: FlowDensity-based gating was applied to 102 normal bone marrow samples and compared with the manual analysis. Independent expression of each cell marker was assessed for comprehensive expression analysis and visualization. Results: Our findings showed a correlation between the manual and flowDensity-based gating in the lymphocyte subsets. However, flowDensity-based gating in the populations with a small number of cells in each cluster showed a low degree of correlation. Comprehensive expression analysis successfully identified and visualized the lymphocyte subsets. Discussion: Our study found that although flowDensity might be a promising method for FCM data analysis, more optimization is required before implementing this algorithm into day-to-day workflow.

Chronic airways infection with methicillin-resistant Staphylococcus aureus (MRSA) is associated with worse respiratory disease cystic fibrosis (CF) patients. Ceftaroline is a cephalosporin that inhibits the penicillin-binding protein (PBP2a) uniquely produced by MRSA. We analyzed 335 S. aureus isolates from CF sputum samples collected at three US centers between 2015–2018. Molecular relationships demonstrated that high-level resistance of preceding isolates to carbapenems were associated with subsequent isolation of ceftaroline resistant CF MRSA. In vitro evolution experiments showed that pre-exposure of CF MRSA to meropenem with further selection with ceftaroline implied mutations in mecA and additional mutations in pbp1 and pbp2 , targets of carbapenems; no effects were achieved by other β-lactams. An in vivo pneumonia mouse model showed the potential therapeutic efficacy of ceftaroline/meropenem combination against ceftaroline-resistant CF MRSA infections. Thus, the present findings highlight risk factors and potential therapeutic strategies offering an opportunity to both prevent and address antibiotic resistance in this patient population. Varela, Roch et al. find that resistance of preceding isolates to carbapenems is associated with the subsequent isolation of methicillin-resistant Staphylococcus aureus (MRSA) resistant to ceftaroline. Addition of meropenem reduces the cystic fibrosis MRSA’s resistance to ceftaroline in a pneumonia mouse model, suggesting a strategy to control antibiotics resistance.

This study examined the hiring practices of Pennsylvania public school principals and the effect that teacher selection training from principal preparation programs and professional development opportunities has on principal practice. The study also examined the impact of teacher shortages on the teacher selection processes used by principals. The research design for the study was an explanatory sequential mixed methods design. Two hundred twenty-six principals participated in a quantitative survey that presented data regarding the teacher selection strategies used by principals and teacher selection training principals received. The quantitative data was analyzed and used to direct the qualitative phase of the study. Eight principals participated in a follow-up semi-structured interview to further explore teacher selection skill development and the impact of teacher shortages on hiring practices. The significant findings include the teacher selection training principals receive impacts the use of pre-interview screening and identifying critical qualities in a potential teacher, both research-based best practices for hiring. The study also revealed that most principals are not receiving teacher selection training during their principal preparation graduate programs or professional development opportunities, instead relying on unstructured learning opportunities such as reading articles or observing other administrators, creating inconsistency of practice. Finally, the study revealed that principals are experiencing teacher shortages during hiring, resulting in less qualified applicants and lower expectations.

Pathology training programs throughout the United States have endured unprecedented challenges dealing with the ongoing coronavirus disease 2019 pandemic. At Houston Methodist Hospital, the Department of Pathology and Genomic Medicine planned and executed a trainee-oriented, stepwise emergency response. The focus was on optimizing workflows among areas of both clinical and anatomic pathology, maintaining an excellent educational experience, and minimizing trainee exposure to coronavirus disease 2019. During the first phase of the response, trainees were divided into 2 groups: one working on-site and the other working remotely. With the progression of the pandemic, all trainees were called back on-site and further redeployed within our department to meet the significantly increased workload demands of our clinical laboratory services. Adjustments to trainee educational activities included, among others, the organization of a daily coronavirus disease 2019 virtual seminar series. This series served to facilitate communication between faculty, laboratory managers, and trainees. Moreover, it became a forum for trainees to provide updates on individual service workflows and volumes, ongoing projects and research, as well as literature reviews on coronavirus disease 2019–related topics. From our program’s experience, redeploying pathology trainees within our department during the coronavirus disease 2019 pandemic resulted in optimization of patient care while ensuring trainee safety, and importantly, helped to maintain continuous high-quality education through active involvement in unique learning opportunities.

We have produced transgenic maize plants containing a wheat Glu-1Dx5 gene encoding the high-molecular-weight glutenin subunit 1Dx5. Analysis by SDS-PAGE showed that a protein similar in size to the wheat 1Dx5 subunit accumulates in the endosperm of transgenic maize from four independent transformation events. This protein reacts with a monoclonal antibody specific to the wheat 1Dx5 subunit and was not detected in nontransgenic controls or in pollen, anthers, leaves or embryos of plants grown from seeds expressing this protein in endosperm. Genomic Southern-blot analysis is consistent with results from SDS-PAGE and indicates that the transgene integration sites are complex and are different in the four events studied. Using the presence of this protein as a phenotypic marker, we studied the inheritance of this gene through three sexual generations. Reciprocal crosses with nontransgenic plants and self-pollinations were performed, and the resulting kernels were analyzed for the presence of the 1Dx5 subunit. These data, together with PCR analysis for the transgene, suggest that the transgene is inefficiently transmitted through pollen in all four events.

Carry and sum circuits for a 2-bit adder used in a pipelined 2N-bit adder-accumulator architecture are reported. To obtain high clock rates in a design with multiple gate delays a novel merged AND-OR-Latch structure using four-level series gated current steering logic is employed. These integrated circuits were fabricated in InAlAs/InGaAs transferred substrate heterojunction bipolar transistor technology and operate up to 19 GHz clock rate.

Circulating tumor DNA is detected with high sensitivity and specificity using molecular barcoding and in silico error correction. High-throughput sequencing of circulating tumor DNA (ctDNA) promises to facilitate personalized cancer therapy. However, low quantities of cell-free DNA (cfDNA) in the blood and sequencing artifacts currently limit analytical sensitivity. To overcome these limitations, we introduce an approach for integrated digital error suppression (iDES). Our method combines in silico elimination of highly stereotypical background artifacts with a molecular barcoding strategy for the efficient recovery of cfDNA molecules. Individually, these two methods each improve the sensitivity of cancer personalized profiling by deep sequencing (CAPP-Seq) by about threefold, and synergize when combined to yield ∼15-fold improvements. As a result, iDES-enhanced CAPP-Seq facilitates noninvasive variant detection across hundreds of kilobases. Applied to non-small cell lung cancer (NSCLC) patients, our method enabled biopsy-free profiling of EGFR kinase domain mutations with 92% sensitivity and >99.99% specificity at the variant level, and with 90% sensitivity and 96% specificity at the patient level. In addition, our approach allowed monitoring of NSCLC ctDNA down to 4 in 10 5 cfDNA molecules. We anticipate that iDES will aid the noninvasive genotyping and detection of ctDNA in research and clinical settings.

A 75 GHz static frequency divider in InAlAs/InGaAs transferred-substrate heterojunction bipolar transistor (HBT) technology is reported. This is the highest reported frequency of operation for a static frequency divider. The circuit has 60 transistors and dissipates 800 mW. The divider was operated at a clock frequency of 5.0 to 75 GHz.

Aaron Newman and his colleagues introduce a next-generation sequencing–based approach for the cost-effective detection and quantitation of tumor-derived circulating DNA in both early- and advanced-stage tumors and with high levels of sensitivity and specificity. CAPP-Seq (cancer personalized profiling by deep sequencing) can simultaneously detect multiple mutations and mutation types, including rearrangements. Here, utility is demonstrated for non–small-cell lung cancer. Circulating tumor DNA (ctDNA) is a promising biomarker for noninvasive assessment of cancer burden, but existing ctDNA detection methods have insufficient sensitivity or patient coverage for broad clinical applicability. Here we introduce cancer personalized profiling by deep sequencing (CAPP-Seq), an economical and ultrasensitive method for quantifying ctDNA. We implemented CAPP-Seq for non–small-cell lung cancer (NSCLC) with a design covering multiple classes of somatic alterations that identified mutations in >95% of tumors. We detected ctDNA in 100% of patients with stage II–IV NSCLC and in 50% of patients with stage I, with 96% specificity for mutant allele fractions down to ∼0.02%. Levels of ctDNA were highly correlated with tumor volume and distinguished between residual disease and treatment-related imaging changes, and measurement of ctDNA levels allowed for earlier response assessment than radiographic approaches. Finally, we evaluated biopsy-free tumor screening and genotyping with CAPP-Seq. We envision that CAPP-Seq could be routinely applied clinically to detect and monitor diverse malignancies, thus facilitating personalized cancer therapy.