Effect of activation rate on output torque values from a dental implant toggle-style torque wrench.

STATEMENT OF PROBLEM: The output torque delivered by a dental implant toggle-style torque wrench is known to be affected by activation rate. The International Organization for Standardization (ISO) established the ISO 6789-1 standard to provide guidance on activation rates relative to desired output torque in the Nm torque range. Whether the ISO 6789-1 standard applies at the relatively lower dental torque ranges is not known, and little information is available on the activation rates that clinicians use and how this may affect output torque. PURPOSE: The purpose of this in vitro study was to determine how output torque values vary with the activation rates used by clinicians at dental implant-relevant target torque values. MATERIAL AND METHODS: To determine clinically relevant activation rates, a new adjustable dental implant toggle-style torque wrench was activated from 0 to 25 Ncm target torque by 5 prosthodontists by using a custom mandibular and maxillary typodont model containing implants and abutments with screws. This provided a baseline of activation rates (mm/sec). Data were transferred to a computerized numerical control model incorporating a variable speed linear motor, which was used to drive a dental implant toggle-style torque wrench attached to an electronic torque measuring device. Constant speed and a regulated dual-speed-assigned 80/20 rate group, where 80% of target torque value was delivered first, then a pause, and the final 20% at different speeds as suggested by the ISO 6789-1 standard, were evaluated. Fast, medium, and slow rates were categorized and applied with target torque values of 10, 25, and 35 Ncm, respectively, for n=12 activations. The output torque values were recorded for both constant and 80/20 groups and compared with the desired target torque values. Data were statistically analyzed with 1-way ANOVA and the Scheffé post hoc paired t test (α=.05). RESULTS: The clinicians' activation rates from 0 to 25 Ncm on the typodont model converted into linear speeds resulted in fast =24.19 mm/sec, medium =14.5 mm/sec, and slow =7.25 mm/sec. When actioned at a constant rate, the mean output torque values were generally in the order of slow > medium > fast in activation rates. Generally, precision output torque decreased as target torque increased, especially when slow and medium rates, either constant or regulated 80/20 were used. All mean output torque for slow, medium, and their 80/20 variants were greater than target torque. Fast and 80/20 fast produced the lowest mean output torque values for all torque settings, and at the 35 Ncm setting, the dental implant toggle-style torque wrench output torque mean values were lower than target torque. Statistically significant differences (P<.05) were found among groups, most notably in the 80/20 dual-speed groups, especially in comparisons with the 80/20 fast rate group. When the target torque value was 10 Ncm with the 80/20 fast rate, the output torque value deviated from the maximum ISO limit by more than 6%. CONCLUSIONS: The rate of actioning a dental implant toggle-style torque wrench influenced the delivered torque value, with fast rate actioning producing lower and less precise output torque values.

Blockade of Discoidin Domain Receptor Signaling with Sitravatinib Reveals DDR2 as a Mediator of Neuroblastoma Pathogenesis and Metastasis.

Oncogene-driven expression and activation of receptor tyrosine kinases (RTK) promotes tumorigenesis and contributes to drug resistance. Increased expression of the kinases DDR2 (Discoid Domain Receptor 2), RET, PDGFRA, KIT, MET, and ALK (Anaplastic Lymphoma Kinase) independently correlate with decreased overall survival (OS) and event free survival (EFS) of pediatric neuroblastoma. The multikinase inhibitor sitravatinib targets DDR2, RET, PDGFRA, KIT and MET with low nanomolar activity and we therefore tested its efficacy against orthotopic and syngeneic tumor models. Sitravatinib markedly reduced cell proliferation and migration in vitro independently of MYCN (N-Myc proto-oncogene), ALK, or MYC (c-Myc proto-oncogene) status, and inhibited proliferation and metastasis of human orthotopic xenografts. Oral administration of sitravatinib to homozygous Th-MYCN transgenic mice (Th-MYCN+/+) after tumor initiation completely arrested further tumor development with no mice dying of disease while maintained on sitravatinib treatment (control cohort 57 days median time to sacrifice). Among these top kinases, DDR2 expression has the strongest correlation with poor survival and high stage at diagnosis, and the highest sensitivity to the drug. We confirmed on-target inhibition of collagen-mediated activation of DDR2. Genetic knockdown of DDR2 partially phenocopies Sitravatinib treatment, limiting tumor development and metastasis across tumor models. Analysis of single cell sequencing data demonstrated that DDR2 is restricted to mesenchymal-type tumor subpopulations and is enriched in Schwann Cell Precursor (SCP) subpopulations found in high-risk disease. These data define an unsuspected role for sitravatinib as a therapeutic agent in neuroblastoma and reveal a novel function for DDR2 as a driver of tumor growth and metastasis.

Pigment cell progenitor heterogeneity and reiteration of developmental signaling underlie melanocyte regeneration in zebrafish.

Tissue-resident stem and progenitor cells are present in many adult organs, where they are important for organ homeostasis and repair in response to injury. However, the signals that activate these cells and the mechanisms governing how these cells renew or differentiate are highly context-dependent and incompletely understood, particularly in non-hematopoietic tissues. In the skin, melanocyte stem and progenitor cells are responsible for replenishing mature pigmented melanocytes. In mammals, these cells reside in the hair follicle bulge and bulb niches where they are activated during homeostatic hair follicle turnover and following melanocyte destruction, as occurs in vitiligo and other skin hypopigmentation disorders. Recently, we identified melanocyte progenitors in adult zebrafish skin. To elucidate mechanisms governing melanocyte progenitor renewal and differentiation we analyzed individual transcriptomes from thousands of melanocyte lineage cells during the regeneration process. We identified transcriptional signatures for progenitors, deciphered transcriptional changes and intermediate cell states during regeneration, and analyzed cell-cell signaling changes to discover mechanisms governing melanocyte regeneration. We identified KIT signaling via the RAS/MAPK pathway as a regulator of melanocyte progenitor direct differentiation and asymmetric division. Our findings show how activation of different subpopulations of mitfa-positive cells underlies cellular transitions required to properly reconstitute the melanocyte pigmentary system following injury.

Microfluidic pumps for cell sorting.

Microfluidic cell sorting has shown promising advantages over traditional bulky cell sorting equipment and has demonstrated wide-reaching applications in biological research and medical diagnostics. The most important characteristics of a microfluidic cell sorter are its throughput, ease of use, and integration of peripheral equipment onto the chip itself. In this review, we discuss the six most common methods for pumping fluid samples in microfluidic cell sorting devices, present their advantages and drawbacks, and discuss notable examples of their use. Syringe pumps are the most commonly used method for fluid actuation in microfluidic devices because they are easily accessible but they are typically too bulky for portable applications, and they may produce unfavorable flow characteristics. Peristaltic pumps, both on- and off-chip, can produce reversible flow but they suffer from pulsatile flow characteristics, which may not be preferable in many scenarios. Gravity-driven pumping, and similarly hydrostatic pumping, require no energy input but generally produce low throughputs. Centrifugal flow is used to sort cells on the basis of size or density but requires a large external rotor to produce centrifugal force. Electroosmotic pumping is appealing because of its compact size but the high voltages required for fluid flow may be incompatible with live cells. Emerging methods with potential for applications in cell sorting are also discussed. In the future, microfluidic cell sorting methods will trend toward highly integrated systems with high throughputs and low sample volume requirements.

The Tumor-Associated Calcium Signal Transducer 2 (TACSTD2) oncogene is upregulated in pre-cystic epithelial cells revealing a new target for polycystic kidney disease.

Polycystic kidney disease (PKD) is an important cause of end stage renal disease, but treatment options are limited. While later stages of the disease have been extensively studied, mechanisms driving the initial conversion of renal tubules into cysts are not understood. To identify factors that promote the initiation of cysts we deleted polycystin-2 ( Pkd2 ) in mice and surveyed transcriptional changes before and immediately after cysts developed. We identified 74 genes which we term cyst initiation candidates (CICs). To identify conserved changes with relevance to human disease we compared these murine CICs to single cell transcriptomic data derived from patients with PKD and from healthy controls. Tumor-associated calcium signal transducer 2 ( Tacstd2 ) stood out as an epithelial-expressed gene whose levels were elevated prior to cystic transformation and further increased with disease progression. Human tissue biopsies and organoids show that TACSTD2 protein is low in normal kidney cells but is elevated in cyst lining cells. While TACSTD2 has not been studied in PKD, it has been studied in cancer where it is highly expressed in solid tumors while showing minimal expression in normal tissue. This property is being exploited by antibody drug conjugates that target TACSTD2 for the delivery of cytotoxic drugs. Our finding that Tacstd2 is highly expressed in cysts, but not normal tissue, suggests that it should be explored as a candidate for drug development in PKD. More immediately, our work suggests that PKD patients undergoing TACSTD2 treatment for cancer should be monitored for kidney effects. One Sentence Summary: The oncogene, tumor-associated calcium signal transducer 2 (Tacstd2) mRNA increased in abundance shortly after Pkd2 loss and may be a driver of cyst initiation in polycystic kidney disease.

Thermophobic Trehalose Glycopolymers as Smart C-Type Lectin Receptor Vaccine Adjuvants.

Herein, this work reports the first synthetic vaccine adjuvants that attenuate potency in response to small, 1-2 °C changes in temperature about their lower critical solution temperature (LCST). Adjuvant additives significantly increase vaccine efficacy. However, adjuvants also cause inflammatory side effects, such as pyrexia, which currently limits their use. To address this, a thermophobic vaccine adjuvant engineered to attenuate potency at temperatures correlating to pyrexia is created. Thermophobic adjuvants are synthesized by combining a rationally designed trehalose glycolipid vaccine adjuvant with thermoresponsive poly-N-isoporpylacrylamide (NIPAM) via reversible addition fragmentation chain transfer (RAFT) polymerization. The resulting thermophobic adjuvants exhibit LCSTs near 37 °C, and self-assembled into nanoparticles with temperature-dependent sizes (90-270 nm). Thermophobic adjuvants activate HEK-mMINCLE and other innate immune cell lines as well as primary mouse bone marrow derived dendritic cells (BMDCs) and bone marrow derived macrophages (BMDMs). Inflammatory cytokine production is attenuated under conditions mimicking pyrexia (above the LCST) relative to homeostasis (37 °C) or below the LCST. This thermophobic behavior correlated with decreased adjuvant Rg is observed by DLS, as well as glycolipid-NIPAM shielding interactions are observed by NOESY-NMR. In vivo, thermophobic adjuvants enhance efficacy of a whole inactivated influenza A/California/04/2009 virus vaccine, by increasing neutralizing antibody titers and CD4+ /44+ /62L+ lung and lymph node central memory T cells, as well as providing better protection from morbidity after viral challenge relative to unadjuvanted control vaccine. Together, these results demonstrate the first adjuvants with potency regulated by temperature. This work envisions that with further investigation, this approach can enhance vaccine efficacy while maintaining safety.

Research Techniques Made Simple: Zebrafish Models for Human Dermatologic Disease.

Skin diseases affect nearly one third of the world's population. Disease types range from oncologic to inflammatory, and outcomes can be as severe as death and disfigurement. Although many skin diseases have been modeled in murine models, the advantages of zebrafish models have led to recent increasing use in modeling human disease. Their rapid development, comparable skin architecture, tractable genetics, unparalleled optical properties, and straightforward drug screens make them an excellent model to study skin disease. In this review, we discuss the attributes of the zebrafish model system as well as current zebrafish models for dermatologic diseases, including melanoma, squamous cell carcinoma, vitiligo, epidermal bullosa, psoriasis, and wounding.

Working together: Heterotypic clusters and collective cell migration in melanoma metastasis.

In this issue of Developmental Cell, Campbell et al. (2021) show that melanoma cells with distinct invasive or proliferative gene signatures can form heterotypic clusters that extravasate collectively and readily seed the growth of metastatic lesions. These findings highlight interactions between heterogenous tumor cells as being critical for metastasis.

From Tank to Treatment: Modeling Melanoma in Zebrafish.

Melanoma is the deadliest form of skin cancer and one of few cancers with a growing incidence. A thorough understanding of its pathogenesis is fundamental to developing new strategies to combat mortality and morbidity. Zebrafish-due in large part to their tractable genetics, conserved pathways, and optical properties-have emerged as an excellent system to model melanoma. Zebrafish have been used to study melanoma from a single tumor initiating cell, through metastasis, remission, and finally into relapse. In this review, we examine seminal zebrafish studies that have advanced our understanding of melanoma.