Uncovering Minimal Pathways in Melanoma Initiation.

Cutaneous melanomas are clinically and histologically heterogeneous. Most display activating mutations in Braf or Nras and complete loss of function of one or more tumor suppressor genes. Mouse models that replicate such mutations produce fast-growing, pigmented tumors. However, mice that combine Braf activation with only heterozygous loss of Pten also produce tumors and, as we show here, in an Albino background this occurs even with Braf activation alone. Such tumors arise rarely, grow slowly, and express low levels of pigmentation genes. The timing of their appearance was consistent with a single step stochastic event, but no evidence could be found that it required de novo mutation, suggesting instead the involvement of an epigenetic transition. Single-cell transcriptomic analysis revealed such tumors to be heterogeneous, including a minor cell type we term LNM ( L ow-pigment, N eural- and extracellular M atrix-signature) that displays gene expression resembling "neural crest"-like cell subsets detected in the fast-growing tumors of more heavily-mutated mice, as well as in human biopsy and xenograft samples. We provide evidence that LNM cells pre-exist in normal skin, are expanded by Braf activation, can transition into malignant cells, and persist with malignant cells through multiple rounds of transplantation. We discuss the possibility that LNM cells not only serve as a pre-malignant state in the production of some melanomas, but also as an important intermediate in the development of drug resistance.

Signalling by senescent melanocytes hyperactivates hair growth.

Niche signals maintain stem cells in a prolonged quiescence or transiently activate them for proper regeneration1. Altering balanced niche signalling can lead to regenerative disorders. Melanocytic skin nevi in human often display excessive hair growth, suggesting hair stem cell hyperactivity. Here, using genetic mouse models of nevi2,3, we show that dermal clusters of senescent melanocytes drive epithelial hair stem cells to exit quiescence and change their transcriptome and composition, potently enhancing hair renewal. Nevus melanocytes activate a distinct secretome, enriched for signalling factors. Osteopontin, the leading nevus signalling factor, is both necessary and sufficient to induce hair growth. Injection of osteopontin or its genetic overexpression is sufficient to induce robust hair growth in mice, whereas germline and conditional deletions of either osteopontin or CD44, its cognate receptor on epithelial hair cells, rescue enhanced hair growth induced by dermal nevus melanocytes. Osteopontin is overexpressed in human hairy nevi, and it stimulates new growth of human hair follicles. Although broad accumulation of senescent cells, such as upon ageing or genotoxic stress, is detrimental for the regenerative capacity of tissue4, we show that signalling by senescent cell clusters can potently enhance the activity of adjacent intact stem cells and stimulate tissue renewal. This finding identifies senescent cells and their secretome as an attractive therapeutic target in regenerative disorders.

Development and validation of a high throughput SARS-CoV-2 whole genome sequencing workflow in a clinical laboratory.

Monitoring new mutations in SARS-CoV-2 provides crucial information for identifying diagnostic and therapeutic targets and important insights to achieve a more effective COVID-19 control strategy. Next generation sequencing (NGS) technologies have been widely used for whole genome sequencing (WGS) of SARS-CoV-2. While various NGS methods have been reported, one chief limitation has been the complexity of the workflow, limiting the scalability. Here, we overcome this limitation by designing a laboratory workflow optimized for high-throughput studies. The workflow utilizes modified ARTIC network v3 primers for SARS-CoV-2 whole genome amplification. NGS libraries were prepared by a 2-step PCR method, similar to a previously reported tailed PCR method, with further optimizations to improve amplicon balance, to minimize amplicon dropout for viral genomes harboring primer-binding site mutation(s), and to integrate robotic liquid handlers. Validation studies demonstrated that the optimized workflow can process up to 2688 samples in a single sequencing run without compromising sensitivity and accuracy and with fewer amplicon dropout events compared to the standard ARTIC protocol. We additionally report results for over 65,000 SARS-CoV-2 whole genome sequences from clinical specimens collected in the United States between January and September of 2021, as part of an ongoing national genomics surveillance effort.

Biological control of incrusting organisms and sediments in Chilean oyster cultures / Controle biológico dos organismos incrustáveis e sedimentos em culturas de ostras Chilenas

The oyster culture has the incrusting organism as problem for production, in this context, it evaluated as biological control against incrusting organism and sediments the introduction of gastropod Tegula atra (Lesson, 1830) in Chilean oysters (Triostrea chilensis Phillippi, 1844) cultures in conditions of starvation presence and absence located in floating cages and bottom cultures. The predation and mechanic effect on T. atra grazing generated a decreasing in seven days of 19.8% and 13.7% of incrusting organisms in cage culture and bottom sediments by effects of gastropods without starvation respectively. Whereas it had a decrease of 12.6% and 11.4% of incrusting organisms in cage culture and bottom sediments by effects of gastropods with starvation respectively. The incrusting organism removed were mainly algae, colonial ascidia, polychaeta, bryozoan and small crustaceans.

A cultura da ostra tem como problema de produção o organismo incrustante, neste contexto, avaliou como controle biológico contra organismos incrustantes e sedimentos a introdução do gastrópode Tegula atra (Lesson, 1830) em culturas de ostras chilenas (Triostrea chilensis Phillippi, 1844) em condições de presença e ausência de fome, localizadas em gaiolas flutuantes e culturas de fundo. A predação e o efeito mecânico no pastejo de T. atra geraram uma diminuição em sete dias de 19,8% e 13,7% dos organismos incrustantes na cultura em gaiola e nos sedimentos de fundo, por efeito de gastrópodes sem fome, respectivamente. Considerando que houve decréscimo de 12,6% e 11,4% dos organismos incrustantes na cultura em gaiola e nos sedimentos de fundo pelos efeitos dos gastrópodes com fome respectivamente. Os organismos incrustantes removidos eram principalmente algas, ascídias coloniais, poliquetas, briozoários e pequenos crustáceos.

Biological control of incrusting organisms and sediments in Chilean oyster cultures / Controle biológico dos organismos incrustáveis e sedimentos em culturas de ostras Chilenas

The oyster culture has the incrusting organism as problem for production, in this context, it evaluated as biological control against incrusting organism and sediments the introduction of gastropod Tegula atra (Lesson, 1830) in Chilean oysters (Triostrea chilensis Phillippi, 1844) cultures in conditions of starvation presence and absence located in floating cages and bottom cultures. The predation and mechanic effect on T. atra grazing generated a decreasing in seven days of 19.8% and 13.7% of incrusting organisms in cage culture and bottom sediments by effects of gastropods without starvation respectively. Whereas it had a decrease of 12.6% and 11.4% of incrusting organisms in cage culture and bottom sediments by effects of gastropods with starvation respectively. The incrusting organism removed were mainly algae, colonial ascidia, polychaeta, bryozoan and small crustaceans.

A cultura da ostra tem como problema de produção o organismo incrustante, neste contexto, avaliou como controle biológico contra organismos incrustantes e sedimentos a introdução do gastrópode Tegula atra (Lesson, 1830) em culturas de ostras chilenas (Triostrea chilensis Phillippi, 1844) em condições de presença e ausência de fome, localizadas em gaiolas flutuantes e culturas de fundo. A predação e o efeito mecânico no pastejo de T. atra geraram uma diminuição em sete dias de 19,8% e 13,7% dos organismos incrustantes na cultura em gaiola e nos sedimentos de fundo, por efeito de gastrópodes sem fome, respectivamente. Considerando que houve decréscimo de 12,6% e 11,4% dos organismos incrustantes na cultura em gaiola e nos sedimentos de fundo pelos efeitos dos gastrópodes com fome respectivamente. Os organismos incrustantes removidos eram principalmente algas, ascídias coloniais, poliquetas, briozoários e pequenos crustáceos.

Biological control of incrusting organisms and sediments in Chilean oyster cultures

Abstract The oyster culture has the incrusting organism as problem for production, in this context, it evaluated as biological control against incrusting organism and sediments the introduction of gastropod Tegula atra (Lesson, 1830) in Chilean oysters (Triostrea chilensis Phillippi, 1844) cultures in conditions of starvation presence and absence located in floating cages and bottom cultures. The predation and mechanic effect on T. atra grazing generated a decreasing in seven days of 19.8% and 13.7% of incrusting organisms in cage culture and bottom sediments by effects of gastropods without starvation respectively. Whereas it had a decrease of 12.6% and 11.4% of incrusting organisms in cage culture and bottom sediments by effects of gastropods with starvation respectively. The incrusting organism removed were mainly algae, colonial ascidia, polychaeta, bryozoan and small crustaceans.

Resumo A cultura da ostra tem como problema de produção o organismo incrustante, neste contexto, avaliou como controle biológico contra organismos incrustantes e sedimentos a introdução do gastrópode Tegula atra (Lesson, 1830) em culturas de ostras chilenas (Triostrea chilensis Phillippi, 1844) em condições de presença e ausência de fome, localizadas em gaiolas flutuantes e culturas de fundo. A predação e o efeito mecânico no pastejo de T. atra geraram uma diminuição em sete dias de 19,8% e 13,7% dos organismos incrustantes na cultura em gaiola e nos sedimentos de fundo, por efeito de gastrópodes sem fome, respectivamente. Considerando que houve decréscimo de 12,6% e 11,4% dos organismos incrustantes na cultura em gaiola e nos sedimentos de fundo pelos efeitos dos gastrópodes com fome respectivamente. Os organismos incrustantes removidos eram principalmente algas, ascídias coloniais, poliquetas, briozoários e pequenos crustáceos.

Biological control of incrusting organisms and sediments in Chilean oyster cultures.

The oyster culture has the incrusting organism as problem for production, in this context, it evaluated as biological control against incrusting organism and sediments the introduction of gastropod Tegula atra (Lesson, 1830) in Chilean oysters (Triostrea chilensis Phillippi, 1844) cultures in conditions of starvation presence and absence located in floating cages and bottom cultures. The predation and mechanic effect on T. atra grazing generated a decreasing in seven days of 19.8% and 13.7% of incrusting organisms in cage culture and bottom sediments by effects of gastropods without starvation respectively. Whereas it had a decrease of 12.6% and 11.4% of incrusting organisms in cage culture and bottom sediments by effects of gastropods with starvation respectively. The incrusting organism removed were mainly algae, colonial ascidia, polychaeta, bryozoan and small crustaceans.

Dynamics of nevus development implicate cell cooperation in the growth arrest of transformed melanocytes.

Mutational activation of the BRAF proto-oncogene in melanocytes reliably produces benign nevi (pigmented 'moles'), yet the same change is the most common driver mutation in melanoma. The reason nevi stop growing, and do not progress to melanoma, is widely attributed to a cell-autonomous process of 'oncogene-induced senescence'. Using a mouse model of Braf-driven nevus formation, analyzing both proliferative dynamics and single-cell gene expression, we found no evidence that nevus cells are senescent, either compared with other skin cells, or other melanocytes. We also found that nevus size distributions could not be fit by any simple cell-autonomous model of growth arrest, yet were easily fit by models based on collective cell behavior, for example in which arresting cells release an arrest-promoting factor. We suggest that nevus growth arrest is more likely related to the cell interactions that mediate size control in normal tissues, than to any cell-autonomous, 'oncogene-induced' program of senescence.

Melanocytes are pigment-producing cells found throughout the skin. Mutations that activate a gene called BRAF cause these cells to divide and produce melanocytic nevi, also known as "moles". These mutations are oncogenic, meaning they can cause cancer. Indeed, BRAF is the most commonly mutated gene in melanoma, a deadly skin cancer that arises from melanocytes. Yet, moles hardly ever progress to melanoma. A proposed explanation for this behavior is that, once activated, BRAF initiates a process called "oncogene-induced senescence" in each melanocyte. This process, likened to premature aging, is thought to be what causes cells in a mole to quit dividing. Although this hypothesis is widely accepted, it has proved difficult to test directly. To investigate this notion, Ruiz-Vega et al. studied mice with hundreds of moles created by the same BRAF mutation found in human moles. Analyzing the activity of genes in individual cells revealed that nevus melanocytes that have stopped growing are no more senescent than other skin cells, including non-mole melanocytes. Ruiz-Vega et al. then analyzed the sizes at which moles stopped growing, estimating the number of cells in each mole. The data were then compared with the results of a simulation and mathematical modeling. This revealed that any model based on the idea of cells independently shutting down after a number of random events could not reproduce the distribution of mole sizes that had been experimentally observed. On the other hand, models based on melanocytes acting collectively to shut down each other's growth fit the observed data much better. These findings suggest that moles do not stop growing as a direct result of the activation of BRAF, but because they sense and respond to their own overgrowth. The same kind of collective sensing is observed in normal tissues that maintain a constant size. Discovering that melanocytes do this not only sheds light on why moles stop growing, it could also help researchers devise new ways to prevent melanomas from forming.

In-Hospital Complications and Readmission in Patients with Hemophilia Undergoing Hip or Knee Arthroplasty.

BACKGROUND: Individuals with hemophilia undergoing hip or knee arthroplasty are at risk for complications such as bleeding and infection. However, data on hospital length of stay (LOS) and readmission rates compared with nonhemophilic controls are lacking. This study compared the complication rates, LOS, and unplanned 30-day readmission rates between patients with hemophilia and nonhemophilic controls. METHODS: This retrospective cohort study used the Pennsylvania Health Care Cost Containment Council (PHC4) database from 2007 to 2015 to compare outcomes in patients with hemophilia and nonhemophilic controls undergoing partial and total hip arthroplasty, knee arthroplasty, and revision knee arthroplasty. RESULTS: A total of 118 patients with hemophilia and 3,811 controls were identified. Compared with controls, patients with hemophilia had a higher risk of bleeding complications after hip procedures (38.7% versus 16.1%, p = 0.003), a higher risk of surgical site infection after knee procedures (8.1% versus 1.1%, p < 0.001), longer median LOS after hip (6 versus 3 days, p < 0.001) and knee (5 versus 3 days, p < 0.001) procedures, and higher rates of unplanned 30-day readmission after hip (22.6% versus 4.1%, p < 0.001) and knee (10.3% versus 4.5%, p = 0.018) procedures. The most common reason for unplanned 30-day readmission in patients with hemophilia was bleeding or the patient's underlying coagulopathy (25.1%). CONCLUSIONS: Patients with hemophilia undergoing hip or knee arthroplasty had a higher incidence of postoperative bleeding (hip procedures) and surgical site infections (knee procedures), longer LOS, and higher rates of unplanned 30-day readmission compared with nonhemophilic controls. Key limitations of our study include the potential for inaccurate coding, the relatively small number of patients in the hemophilia cohort, and the uneven distribution of procedure type in the hemophilia and control cohorts. LEVEL OF EVIDENCE: Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Delineating the role of MITF isoforms in pigmentation and tissue homeostasis.

MITF, a gene that is mutated in familial melanoma and Waardenburg syndrome, encodes multiple isoforms expressed from alternative promoters that share common coding exons but have unique amino termini. It is not completely understood how these isoforms influence pigmentation in different tissues and how the expression of these independent isoforms of MITF is regulated. Here, we show that melanocytes express two isoforms of MITF, MITF-A and MITF-M. The expression of MITF-A is partially regulated by a newly identified retinoid enhancer element located upstream of the MITF-A promoter. Mitf-A knockout mice have only subtle changes in melanin accumulation in the hair and reduced Tyr expression in the eye. In contrast, Mitf-M-null mice have enlarged kidneys, lack neural crest-derived melanocytes in the skin, choroid, and iris stroma, yet maintain pigmentation within the retinal pigment epithelium and iris pigment epithelium of the eye. Taken together, these studies identify a critical role for MITF-M in melanocytes, a minor role for MITF-A in regulating pigmentation in the hair and Tyr expression in the eye, and a novel role for MITF-M in size control of the kidney.

High-resolution melting of the cytochrome B gene in fecal DNA: A powerful approach for fox species identification of the Lycalopex genus in Chile.

Easy, economic, precise species authentication is currently necessary in many areas of research and diagnosis in molecular biology applied to conservation studies of endangered species. Here, we present a new method for the identification of three fox species of the Lycalopex genus in Chile. We developed an assay based on high-resolution melt analysis of the mitochondrial cytochrome B gene, allowing a simple, low cost, fast, and accurate species determination. To validate the assay applicability for noninvasive samples, we collected fecal samples in the Atacama Desert, finding unexpectedly one species outside of its known distribution range. We conclude that the assay has a potential to become a valuable tool for a standardized genetic monitoring of the Lycalopex species in Chile.

The risk of major bleeding in patients with suspected heparin-induced thrombocytopenia.

BACKGROUND: The presence of a hypercoagulable disorder such as heparin-induced thrombocytopenia (HIT) may protect against anticoagulant-associated bleeding. OBJECTIVES: To determine the incidence of major bleeding in patients with suspected HIT. METHODS: We performed a retrospective analysis of 310 patients suspected of having HIT from the Hospital of the University of Pennsylvania and an affiliated community hospital. We compared the cumulative incidence of major bleeding following suspicion for HIT by ultimate HIT status (HIT+ or HIT-) and exposure to an alternative anticoagulant (Tx+ or Tx-). Secondary outcomes included the incidence of new/progressive thrombosis and 30-day mortality. RESULTS: The incidence of major bleeding was high in the HIT+Tx+, HIT- Tx+, and HIT-Tx- groups (35.7%, 44.0%, and 37.3%, respectively). The time to first major bleeding event did not differ between groups (P = .24). Factors associated with increased risk of major bleeding included intensive care unit admission (HR 2.24, 95% CI 1.44-3.47), platelet count < 25 × 109 /L (HR 2.13, 1.10-4.12), and renal dysfunction (HR 1.56, 1.06-2.27); 35.7% of HIT+Tx+, 13.8% HIT-Tx+, and 9.3% of HIT-Tx- patients experienced new or progressive thrombosis. Mortality was similar among the three groups (26.2% HIT+Tx+, 34.5% HIT-Tx+, and 26.7% of HIT-Tx- [P = .34]). CONCLUSIONS: Among patients with suspected HIT, major bleeding was common regardless of HIT status. Contrary to our hypothesis, HIT+ patients were not protected from major bleeding. A better understanding of bleeding risk is needed to inform management decisions in patients with suspected HIT.

Single-cell analysis reveals fibroblast heterogeneity and myeloid-derived adipocyte progenitors in murine skin wounds.

During wound healing in adult mouse skin, hair follicles and then adipocytes regenerate. Adipocytes regenerate from myofibroblasts, a specialized contractile wound fibroblast. Here we study wound fibroblast diversity using single-cell RNA-sequencing. On analysis, wound fibroblasts group into twelve clusters. Pseudotime and RNA velocity analyses reveal that some clusters likely represent consecutive differentiation states toward a contractile phenotype, while others appear to represent distinct fibroblast lineages. One subset of fibroblasts expresses hematopoietic markers, suggesting their myeloid origin. We validate this finding using single-cell western blot and single-cell RNA-sequencing on genetically labeled myofibroblasts. Using bone marrow transplantation and Cre recombinase-based lineage tracing experiments, we rule out cell fusion events and confirm that hematopoietic lineage cells give rise to a subset of myofibroblasts and rare regenerated adipocytes. In conclusion, our study reveals that wounding induces a high degree of heterogeneity among fibroblasts and recruits highly plastic myeloid cells that contribute to adipocyte regeneration.

Prospective comparison of the HEP score and 4Ts score for the diagnosis of heparin-induced thrombocytopenia.

The HIT Expert Probability (HEP) score compared favorably with the 4Ts score in a retrospective study. We assessed the diagnostic accuracy of the HEP score compared with the 4Ts score in a prospective cohort of 310 patients with suspected heparin-induced thrombocytopenia (HIT). A member of the clinical team calculated the HEP score and 4Ts score. An independent panel adjudicated HIT status based on a clinical summary as well as the results of HIT laboratory testing. The prevalence of HIT in the study population was 14.7%. At a cutoff of ≥3, the HEP score was 95.3% sensitive (95% confidence interval [CI], 84.2-99.4) and 35.7% specific (95% CI, 29.8-42.0) for HIT. A 4Ts score of ≥4 had a sensitivity of 97.7% (95% CI, 86.2-99.8) and specificity of 32.9% (95% CI, 27.2-39.1). The areas under the receiver operating characteristic (ROC) curves (AUCs) for the HEP score and 4Ts score were similar (0.81 [95% CI, 0.74-0.87] vs 0.76 [95% CI, 0.69-0.83]; P = .12). The HEP score exhibited a significantly higher AUC than the 4Ts score in patients in the intensive care unit (ICU) (0.86 vs 0.79; P = .03). Among trainee scorers, the HEP score performed significantly better than the 4Ts score (AUC, 0.80 vs 0.73; P = .03). Our data suggest that either the 4Ts score or the HEP score may be used in clinical practice. The HEP score may be preferable in ICU patients and among less experienced clinicians.

Revascularization strategies and in-hospital management in acute coronary syndromes complicated by hemophilia A or hemophilia B.

: Among adult patients with hemophilia A and hemophilia B the emergent management of acute coronary syndromes (ACSs) is challenging, and exposure to antithrombotic agents and/or revascularization procedures may confer an enhanced risk of bleeding. We sought to identify clinical characteristics and in-hospital outcomes among ACS patients with hemophilia A/hemophilia B, compared with matched noncoagulopathic ACS controls. Case discharges from the Nationwide Inpatient Sample, Healthcare Cost and Utilization Project, Agency for Healthcare Research and Quality (1998-2011) had International Classification of Diseases, 9th Revision codes for hemophilia A/hemophilia B and ACS. Control discharges were matched to cases by year of discharge and hospital. Discharges in both groups were assessed for cardiovascular risk factors, type of ACS, use of coronary artery bypass grafting, percutaneous coronary intervention (PCI), bare-metal stent and/or drug-eluting stent, bleeding, and death. In total, 237 cases and 148 848 matched controls were identified. Among cases, HIV/Hepatitis C positivity was more common and obesity/hyperlipidemia less common. ST-elevation myocardial infarction (STEMI) occurred less frequently among hemophilia A cases than controls. hemophilia A and hemophilia B cases were more likely to be managed medically. Cases treated with coronary stent placement were more likely to receive a bare-metal stent than controls. Among PCI, bleeding was more common among hemophilia A cases. The death rates were comparable between groups. ACS-hemophilia A/hemophilia B cases were more often treated noninvasively compared with controls, suggesting an avoidance of PCI/coronary artery bypass grafting in this population, and bleeding (among hemophilia A) was more common. These findings support further study of the management of ACS and in-hospital outcomes among individuals with hemophilia.

ATR Mutations Promote the Growth of Melanoma Tumors by Modulating the Immune Microenvironment.

Melanomas accumulate a high burden of mutations that could potentially generate neoantigens, yet somehow suppress the immune response to facilitate continued growth. In this study, we identify a subset of human melanomas that have loss-of-function mutations in ATR, a kinase that recognizes and repairs UV-induced DNA damage and is required for cellular proliferation. ATR mutant tumors exhibit both the accumulation of multiple mutations and the altered expression of inflammatory genes, resulting in decreased T cell recruitment and increased recruitment of macrophages known to spur tumor invasion. Taken together, these studies identify a mechanism by which melanoma cells modulate the immune microenvironment to promote continued growth.