The bridge helix of Cas12a imparts selectivity in cis-DNA cleavage and regulates trans-DNA cleavage.

Cas12a is an RNA-guided DNA endonuclease of the type V-A CRISPR-Cas system that has evolved convergently with the type II Cas9 protein. We previously showed that proline substitutions in the bridge helix (BH) impart target DNA cleavage selectivity in Streptococcus pyogenes (Spy) Cas9. Here, we examined a BH variant of Cas12a from Francisella novicida (FnoCas12aKD2P ) to test mechanistic conservation. Our results show that for RNA-guided DNA cleavage (cis-activity), FnoCas12aKD2P accumulates nicked products while cleaving supercoiled DNA substrates with mismatches, with certain mismatch positions being more detrimental for linearization. FnoCas12aKD2P also possess reduced trans-single-stranded DNA cleavage activity. These results implicate the BH in substrate selectivity in both cis- and trans-cleavages and show its conserved role in target discrimination among Cas nucleases.

Surface-Treated Pellethanes: Comparative Quantification of Encrustation in Artificial Urine Solution.

Introduction: Encrustation of implanted urinary tract devices is associated with significant morbidity. Pellethane® is a polyether-based compound noted for its strength, porosity, and resistance to solvents. We assessed Pellethane thermoplastic polyurethane (TPU) with and without surface coatings 2-hydroxyethyl methacrylate (HEMA) and tetraethylene glycol dimethyl ether (TETRA) for the potential to resist encrustation in an artificial urine environment. Materials and Methods: Samples of Pellethane TPU, HEMA Pellethane TPU, TETRA Pellethane TPU, and hydrogel-coated ureteral stent (Cook®) were suspended in a batch-flow model with an artificial urine solution (AUS). Every 48 hours for 90 days, 40% of the solution was replaced with fresh AUS. All samples were stored in a 37°C incubator. Subsequently, the samples were thoroughly dried for 48 hours before weighing. Scanning electron microscopy was used to assess the degree of encrustation. Nu-Attom Inductively Coupled Plasma Mass Spectrometry (ICP-MS) was used to determine the precise compositions of the encrustation specifically with regard to calcium, magnesium, and phosphate. Results: At the conclusion of the 90-day trial, the samples were analyzed, and the average mass changes were as follows: stent 63.78%, uncoated Pellethane TPU 11.50%, HEMA-coated Pellethane TPU 2.90%, and TETRA-coated Pellethane TPU 0.60%. Pellethane TPU products, and specifically those coated with HEMA and TETRA, exhibited less average mass increase and a lesser propensity to form encrustation than the traditional urinary tract stent. The mass increases noted on coated Pellethane devices were primarily ionic, whereas that of the stent was not. Conclusion: Pellethane, particularly with an HEMA-based preventative coating, may serve as a favorable alternative to traditional urinary stent material, providing its improved resistance to encrustation.

Evaluation of a point-of-care ultrasound curriculum taught by medical students for physicians, nurses, and midwives in rural Indonesia.

PURPOSE: In the Indonesian health-care system, nurses and midwives often serve as the primary health-care providers due to physician shortages. Seeking to address the need for medical care in resource-limited environments, some have advocated for portable equipment in the hands of health-care providers. We hypothesized that medical students are able to effectively teach point-of-care ultrasound (POCUS) to physicians, nurses, and midwives in rural Indonesia. METHODS: We conducted a prospective, observational study using health-care practitioners from a clinic and accredited school for nursing and midwifery in Mojokerto, East Java, Indonesia. Enrolled practitioners took part in a 4-week POCUS course followed by postinstructional testing. RESULTS: A total of 55 health-care practitioners completed the course. This included 19 physicians, 13 nurses, and 19 midwives. Of the 55 clinicians, 43 (72%) passed the course and 12 (28%) failed. CONCLUSIONS: Physicians, nurses, and midwives in rural Indonesia showed significant acquisition of ultrasound (US) knowledge and skills following a 4-week US course. Following training, all three groups displayed skills in practical US use during a postcourse practical examination. This is one of the first studies to assess the efficacy of medical students teaching POCUS to midwives and nurses.

Energy-Mediated Machinery Drives Cellular Mechanical Allostasis.

Allostasis is a fundamental biological process through which living organisms achieve stability via physiological or behavioral changes to protect against internal and external stresses, and ultimately better adapt to the local environment. However, an full understanding of cellular-level allostasis is far from developed. By employing an integrated micromechanical tool capable of applying controlled mechanical stress on an individual cell and simultaneously reporting dynamic information of subcellular mechanics, individual cell allostasis is observed to occur through a biphasic process; cellular mechanics tends to restore to a stable state through a mechanoadaptative process with excitative biophysical activity followed by a decaying adaptive phase. Based on these observations, it is found that cellular allostasis occurs through a complex balance of subcellular energy and cellular mechanics; upon a transient and local physical stimulation, cells trigger an allostatic state that maximizes energy and overcomes a mechanical "energy barrier" followed by a relaxation state that reaches its mechanobiological stabilization and energy minimization. Discoveries of energy-driven cellular machinery and conserved mechanotransductive pathways underscore the critical role of force-sensitive cytoskeleton equilibrium in cellular allostasis. This highlight the biophysical origin of cellular mechanical allostasis, providing subcellular methods to understand the etiology and progression of certain diseases or aging.

Metastatic neoplasms to the thyroid diagnosed by fine-needle aspiration/core needle biopsy: Clinicopathologic and cytomorphologic correlation.

BACKGROUND: Although thyroid fine-needle aspiration (FNA) and core needle biopsy (CNB) are commonly utilized modalities in the evaluation of thyroid nodules, metastatic tumors to the thyroid are only rarely encountered. We aspired to determine the incidence and primary origin of metastases to the thyroid at our institution and to examine their clinicopathologic and cytomorphologic features. MATERIALS AND METHODS: A search of our database was undertaken to review all thyroid FNA and/or CNB examined between January 2004 and December 2013. RESULTS: During our 10 year study period, 7497 patients underwent 13,182 FNA and/or CNB. Four hundred sixty one (6%) patients were diagnosed with neoplasms. Only five (1.1%) were found to have metastatic tumors to the thyroid involving three females and two males. Two were diagnosed by FNA, one by CNB, and two by both FNA and CNB, with rapid on-site evaluation (ROSE) employed in all cases. The primary malignancies in the five cases were pulmonary and nasopharyngeal squamous cell carcinomas, renal cell carcinoma, pancreatic adenocarcinoma, and olfactory neuroblastoma. The cytomorphologic features of these metastases to the thyroid aided in their distinction from primary thyroid carcinoma. Two of these metastases, a renal cell carcinoma and pancreatic adenocarcinoma, were the first clinical manifestations of cancer. CONCLUSION: Metastases to the thyroid diagnosed by FNA and/or CNB are exceedingly rare in our institution, comprising only 0.04% of total FNA/CNB and only 1.1% of all thyroid neoplasms. We report the first known case of metastatic olfactory neuroblastoma to the thyroid diagnosed by aspiration cytology. In addition, an occult primary may present as a thyroid mass on FNA or CNB as occurred with two of our cases. FNA/CNB proved to be highly effective in the diagnosis of metastases to the thyroid, with ROSE proving valuable in assuring specimen adequacy. Thyroid FNA and CNB demonstrated great utility in the setting of metastatic disease, obviating the need for more invasive procedures.