A very, very lonely, unmagical time. The lived experience of perinatal anxiety: A longitudinal interpretative phenomenological analysis.

PROBLEM: Minimal longitudinal qualitative evidence examining lived experience of anxiety over the perinatal continuum limits holistic understanding of the course of antenatal and postnatal anxiety. BACKGROUND: Perinatal anxiety has deleterious effects on the mother and infant and is more commonly experienced yet less well investigated than perinatal depression. AIM AND METHOD: To explore women's experiences living with perinatal anxiety to increase understanding of the condition; inform support given by midwives and other health professionals and provide practice, education, and research recommendations. Five women were interviewed at three timepoints, producing 15 datasets. Data was analysed using longitudinal interpretative phenomenological analysis. FINDINGS: Nine Group Experiential Themes emerged: the anxious mother, transformation, sets of ears and the anxious pregnancy (antenatal); baby as external focus, returning to oneself and the emotional unknown (early postnatal); and moving on, and shifting sands (late postnatal). Three Longitudinal Experiential Concepts explicated lived experience over time: maternal eyes, transforming existence, and emotional kaleidoscope. The lived experience of perinatal anxiety was revealed as socially constructed, with relationships with self, others, and the world key. The collision between anxiety and motherhood as social constructs provides perinatal anxiety with its unique characteristics. CONCLUSION: Midwives and other healthcare professionals should understand the significance of perinatal anxiety, enabling disclosure of stigmatising and uncomfortable feelings without judgement. Research examining whether perinatal specific screening tools should be used by midwives and exploring the relationship between perinatal anxiety and depression is recommended. Education for clinicians on the significance of perinatal anxiety is essential.

The COVID-19 pandemic's impact on all-cause mortality disparities in Medicare: By race, income, chronic health, mental/behavioral health, disability.

BACKGROUND: The Medicare-enrolled population is heterogeneous across race, ethnicity, age, dual eligibility, and a breadth of chronic health, mental and behavioral health, and disability-related conditions, which may be differentially impacted by the COVID-19 pandemic. OBJECTIVE: To quantify changes in all-cause mortality prior-to and in the first year of the COVID-19 pandemic across Medicare's different sociodemographic and health-condition subpopulations. METHODS: This observational, population-based study used stratified bivariate regression to investigate Medicare fee-for-service subpopulation differences in pre-pandemic (i.e., 2019 versus 2016) and pandemic-related (2020 versus 2019) changes in all-cause mortality. RESULTS: All-cause mortality in the combined Medicare-Advantage (i.e., managed care) and fee-for-service beneficiary population improved by a relative 1% in the ten years that preceded the COVID-19 pandemic, but then escalated by a relative 15.9% in 2020, the pandemic's first year. However, a closer look at Medicare's fee-for-service subpopulations reveals critical differences. All-cause mortality had actually been worsening prior to the pandemic among most psychiatric and disability-related condition groups, all race and ethnicity groups except White Non-Hispanic, and Medicare-Medicaid dual-eligible (i.e., low-income) beneficiaries. Many of these groups then experienced all-cause mortality spikes in 2020 that were over twice that of the overall Medicare fee-for-service population. Of all 61 chronic health conditions studied, beneficiaries with schizophrenia were the most adversely affected, with all-cause mortality increasing 38.4% between 2019 and 2020. CONCLUSION: This analysis reveals subpopulation differences in all-cause mortality trends, both prior to and in year-one of the COVID-19 pandemic, indicating that the events of 2020 exacerbated preexisting health-related inequities.

A road map on synthetic strategies and applications of biodegradable polymers.

Biodegradable polymers have emerged as fascinating materials due to their non-toxicity, environmentally benign nature and good mechanical strength. The toxic effects of non-biodegradable plastics paved way for the development of sustainable and biodegradable polymers. The engineering of biodegradable polymers employing various strategies like radical ring opening polymerization, enzymatic ring opening polymerization, anionic ring opening polymerization, photo-initiated radical polymerization, chemoenzymatic method, enzymatic polymerization, ring opening polymerization and coordinative ring opening polymerization have been discussed in this review. The application of biodegradable polymeric nanoparticles in the biomedical field and cosmetic industry is considered to be an emerging field of interest. However, this review mainly highlights the applications of selected biodegradable polymers like polylactic acid, poly(ε-caprolactone), polyethylene glycol, polyhydroxyalkanoates, poly(lactide-co-glycolide) and polytrimethyl carbonate in various fields like agriculture, biomedical, biosensing, food packaging, automobiles, wastewater treatment, textile and hygiene, cosmetics and electronic devices.

Acquired reactive perforating collagenosis, a rare entity at uncommon site.

Acquired reactive perforating collagenosis (ARPC), rare disorder characterized by transepidermal elimination (TEE) of collagen fibers, is seen in adult diabetics. Genetic predisposition, familial aggregation, trauma, bites and scratching are implicated. Diabetics develop microvascular diseases leading to intense pruritus causing repeated micro trauma leading to necrosis of connective tissue of dermis, causing TEE. Isolated papules, plaques and nodules with central keratotic plugs, are mostly seen on extensor surfaces of limbs but trunk and face may be involved. Histopathology shows extrusion of abnormal collagen fibers through epidermis. Multiple treatment modalities show variable response. A 52 year old diabetic female had multiple, itchy, well defined, erythematous papules and plaques with central adherent crusting on lower back since 1 month. Histopathology showed cup shaped epidermal depression filled with plug of altered collagen, acanthotic epidermis with hyperkeratosis and parakeratosis. Underlying epidermis was thin with fine slits through which vertically oriented basophilic collagen fibers were extruded.

Respiratory Compromise After Anterior Cervical Spine Surgery: Incidence, Subsequent Complications, and Independent Predictors.

STUDY DESIGN: Retrospective cohort study. OBJECTIVE: Respiratory compromise (RC) is a rare but catastrophic complication of anterior cervical spine surgery (ACSS) commonly due to compressive fluid collections or generalized soft tissue swelling in the cervical spine. Established risk factors include operative duration, size of surgical exposure, myelopathy, among others. The purpose of this current study is to identify the incidence and clinical course of patients who develop RC, and identify independent predictors of RC in patients undergoing ACSS for cervical spondylosis. METHODS: A large, prospectively-collected registry was used to identify patients undergoing ACSS for spondylosis. Patients with posterior cervical procedures were excluded. Baseline patient characteristics were compared using bivariate analysis, and multivariate analysis was employed to compare postoperative complications and identify independent predictors of RC. RESULTS: 298 of 52,270 patients developed RC (incidence 0.57%). Patients who developed RC had high rates of 30-day mortality (11.7%) and morbidity (75.8%), with unplanned reoperation and pneumonia the most common. The most common reason for reoperations were hematoma evacuation and tracheostomy. Independent patient-specific factors predictive of RC included increasing patient age, male gender, comorbidities such as chronic cardiac and respiratory disease, preoperative myelopathy, prolonged operative duration, and 2-level ACCFs. CONCLUSION: This is among the largest cohorts of patients to develop RC after ACSS identified to-date and validates a range of independent predictors, many previously only described in case reports. These results are useful for taking preventive measures, identifying high risk patients for preoperative risk stratification, and for surgical co-management discussions with the anesthesiology team.

Hybrid Anterior Cervical Discectomy and Fusion and Cervical Disc Arthroplasty: An Analysis of Short-Term Complications, Reoperations, and Readmissions.

STUDY DESIGN: Retrospective cohort study. OBJECTIVES: Although cervical disc arthroplasty (CDA) has become a well-established and effective treatment for symptomatic cervical degeneration, many patients with multilevel disease are not good candidates for CDA at all levels. For such patients, hybrid surgery (HS)-a combination of adjacent anterior cervical discectomy and fusion (ACDF) and CDA-may be more appropriate. Given the novelty of HS and the relative dearth of studies adequately assessing short-term perioperative complications, this current study sought to assess the short-term morbidity profile of HS, differences in operative duration, length of stay (LOS), and readmission and reoperation rates and reasons relative to a 2-level ACDF cohort. METHODS: All patients who underwent HS and 2-level ACDF were identified between 2011 and 2018 using a large, prospectively collected registry. Baseline patient characteristics and postoperative complications were compared using bivariate and/or multivariate analysis. RESULTS: A total of 390 patients undergoing HS were identified. Two-level procedures were the most common (74.9%). Patients undergoing HS were more likely to be younger, male, and have fewer comorbidities. There were no differences between HS and 2-level ACDF in rates of any postoperative complication, transfusion, readmissions, and operative duration. However, HS had a decreased LOS (0.5 days), relative to a 2-level ACDF. HS patients had low rates of reoperation (1.28%) with 1 case for hematoma evacuation and another for revision CDA. CONCLUSIONS: This study represents one of the largest cohorts of patients undergoing HS reported to date. Patients undergoing HS are not at increased risk of perioperative complications relative to a 2-level ACDF and may benefit from shorter LOS.

"Crippling and unfamiliar": Analysing the concept of perinatal anxiety; definition, recognition and implications for psychological care provision for women during pregnancy and early motherhood.

AIM: To clarify how perinatal anxiety is characterised within the current evidence base and discuss how a clearer definition and understanding of this condition may contribute to improving care provision by midwives and other healthcare professionals. BACKGROUND: Perinatal anxiety is common, occurs more frequently than depression and carries significant morbidity for mother and infant. The concept of perinatal anxiety is ill-defined; this can pose a barrier to understanding, identification and appropriate treatment of the condition. DESIGN: Concept Analysis paper. METHOD: Rodgers' Evolutionary Model of Concept Analysis, with review based on PRISMA principles (see Supplementary File-1). FINDINGS: While somatic presentation of perinatal anxiety shares characteristics with general anxiety, anxiety is a unique condition within the context of the perinatal period. The precursors to perinatal anxiety are grounded in biopsychosocial factors and the sequelae can be significant for mother, foetus, newborn and older child. Due to the unique nature of perinatal anxiety, questions arise about presentation and diagnosis within the context of adjustment to motherhood, whether services meet women's needs and how midwives and other health professionals contribute to this. Most current evidence explores screening tools with little examination of the lived experience of perinatal anxiety. CONCLUSION: Examination of the lived experience of perinatal anxiety is needed to address the gap in evidence and further understand this condition. Service provision should account for the unique nature of the perinatal period and be adapted to meet women's psychological needs at this time, even in cases of mild or moderate distress.

Comparison of effect of curcumin gel and noneugenol periodontal dressing in tissue response, early wound healing, and pain assessment following periodontal flap surgery in chronic periodontitis patients.

BACKGROUND: The study was designed taking into consideration the drawbacks of periodontal dressing and healing properties of curcumin. The aim was to assess and compare the effect of Curcumin gel (Curenext) and noneugenol periodontal dressing (Coe pak) on tissue response, wound healing in the early stages, and pain post periodontal flap surgery in patients diagnosed with chronic periodontitis. MATERIALS AND METHODS: Twenty patients requiring periodontal flap surgery were allotted to two groups at random, one receiving periodontal dressing and the other receiving curcumin for this cross over split-mouth study. Flap surgeries were performed on 2 quadrants with 3 weeks' interval. After suture removal, postoperative sites were assessed for tissue response (tissue color [TC] and tissue edema [TE]) and early wound healing as primary outcomes of the study. The secondary outcome was pain assessment and the number of analgesics taken by the individuals. RESULTS: The two groups showed no significant differences with respect to tissue response, early wound healing, and pain perception. Curcumin group consumed lesser number of analgesics as compared to the one with periodontal dressing. CONCLUSION: It was confirmed that periodontal dressing and curcumin are effective in reducing the TE, normalizing the TC, enhancing the wound healing and reducing the pain perception. Curcumin can thus be used as an alternative to periodontal dressing.

Accidental Strangulation While Playing with Hammock in a Child.

In children, strangulation is a fatal injury due to asphyxia, a terminal event of partial or complete hanging. Homemade hammocks are routinely used as a cradle which is potentially dangerous. We are hereby reporting a 12-year-old female with accidental strangulation occurring as a result of swinging on a hammock made of saree and also in a view to educate the public about the hazards of using homemade hammocks.

Multiparametric profiling of non-small-cell lung cancers reveals distinct immunophenotypes.

BACKGROUND. Immune checkpoint blockade improves survival in a subset of patients with non-small-cell lung cancer (NSCLC), but robust biomarkers that predict response to PD-1 pathway inhibitors are lacking. Furthermore, our understanding of the diversity of the NSCLC tumor immune microenvironment remains limited. METHODS. We performed comprehensive flow cytometric immunoprofiling on both tumor and immune cells from 51 NSCLCs and integrated this analysis with clinical and histopathologic characteristics, next-generation sequencing, mRNA expression, and PD-L1 immunohistochemistry (IHC). RESULTS. Cytometric profiling identified an immunologically "hot" cluster with abundant CD8+ T cells expressing high levels of PD-1 and TIM-3 and an immunologically "cold" cluster with lower relative abundance of CD8+ T cells and expression of inhibitory markers. The "hot" cluster was highly enriched for expression of genes associated with T cell trafficking and cytotoxic function and high PD-L1 expression by IHC. There was no correlation between immunophenotype and KRAS or EGFR mutation, or patient smoking history, but we did observe an enrichment of squamous subtype and tumors with higher mutation burden in the "hot" cluster. Additionally, approximately 20% of cases had high B cell infiltrates with a subset producing IL-10. CONCLUSIONS. Our results support the use of immune-based metrics to study response and resistance to immunotherapy in lung cancer. FUNDING. The Robert A. and Renée E. Belfer Family Foundation, Expect Miracles Foundation, Starr Cancer Consortium, Stand Up to Cancer Foundation, Conquer Cancer Foundation, International Association for the Study of Lung Cancer, National Cancer Institute (R01 CA205150), and the Damon Runyon Cancer Research Foundation.

An Integrative Analysis of the InR/PI3K/Akt Network Identifies the Dynamic Response to Insulin Signaling.

Insulin regulates an essential conserved signaling pathway affecting growth, proliferation, and metabolism. To expand our understanding of the insulin pathway, we combine biochemical, genetic, and computational approaches to build a comprehensive Drosophila InR/PI3K/Akt network. First, we map the dynamic protein-protein interaction network surrounding the insulin core pathway using bait-prey interactions connecting 566 proteins. Combining RNAi screening and phospho-specific antibodies, we find that 47% of interacting proteins affect pathway activity, and, using quantitative phosphoproteomics, we demonstrate that ∼10% of interacting proteins are regulated by insulin stimulation at the level of phosphorylation. Next, we integrate these orthogonal datasets to characterize the structure and dynamics of the insulin network at the level of protein complexes and validate our method by identifying regulatory roles for the Protein Phosphatase 2A (PP2A) and Reptin-Pontin chromatin-remodeling complexes as negative and positive regulators of ribosome biogenesis, respectively. Altogether, our study represents a comprehensive resource for the study of the evolutionary conserved insulin network.

Adaptive resistance to therapeutic PD-1 blockade is associated with upregulation of alternative immune checkpoints.

Despite compelling antitumour activity of antibodies targeting the programmed death 1 (PD-1): programmed death ligand 1 (PD-L1) immune checkpoint in lung cancer, resistance to these therapies has increasingly been observed. In this study, to elucidate mechanisms of adaptive resistance, we analyse the tumour immune microenvironment in the context of anti-PD-1 therapy in two fully immunocompetent mouse models of lung adenocarcinoma. In tumours progressing following response to anti-PD-1 therapy, we observe upregulation of alternative immune checkpoints, notably T-cell immunoglobulin mucin-3 (TIM-3), in PD-1 antibody bound T cells and demonstrate a survival advantage with addition of a TIM-3 blocking antibody following failure of PD-1 blockade. Two patients who developed adaptive resistance to anti-PD-1 treatment also show a similar TIM-3 upregulation in blocking antibody-bound T cells at treatment failure. These data suggest that upregulation of TIM-3 and other immune checkpoints may be targetable biomarkers associated with adaptive resistance to PD-1 blockade.

Vitiligo in Children: A Birds Eye View.

Vitiligo in children is a distinct subset of vitiligo and differs from adult vitiligo. Characteristic features include family history of autoimmune or endocrine disease, higher incidence of segmental vitiligo, development of early or premature graying, increased incidence of autoantibodies and poor response to topical PUVA. The exact prevalence of vitiligo in children varies between 0.1-4% of the world population and seems to be higher in India than in other countries and it occurs more frequently in females. Around 12% to 35% of pediatric vitiligo patients have family members with the disease. The most common type of vitiligo in pediatric patients is vitiligo vulgaris, representing 78% of cases. The most commonly associated autoimmune disease is thyroiditis. Phototherapy and topical corticosteroids are the most commonly used treatments for adult vitiligo but are less useful in the pediatric population.

Osmolar regulation of endothelin-1 production by the inner medullary collecting duct.

AIMS: Endothelin-1 (ET-1) is an autocrine inhibitor of collecting duct (CD) Na(+) and water reabsorption. CD ET-1 production is increased by a high salt diet and is important in promoting a natriuretic response. The mechanisms by which a high salt diet enhances CD ET-1 are being uncovered. In particular, elevated tubule fluid flow, as occurs in salt loading, enhances CD ET-1 synthesis. Tubule fluid solute content and interstitial osmolality can also be altered by a high salt diet, however their effect on CD ET-1 alone, or in combination with flow, is poorly understood. MAIN METHODS: ET-1 mRNA production by a mouse inner medullary CD cell line (mIMCD3) in response to changing flow and/or osmolality was assessed. KEY FINDINGS: Flow or hyperosmolality (using NaCl, mannitol or urea) individually caused an ~2-fold increase in ET-1 mRNA, while flow and hyperosmolality together increased ET-1 mRNA by ~14 fold. The hyperosmolality effect alone and the synergistic effect of flow + hyperosmolality was inhibited by chelation of intracellular Ca(2+), however were not altered by blockade of downstream Ca(2+)-signaling pathways (calcineurin or NFATc), inhibition of cellular Ca(2+) entry channels (purinergic receptors or polycystin-2), or blockade of the epithelial Na(+) channel. Inhibition of NFAT5 with rottlerin or NFAT5 siRNA greatly reduced the stimulatory effect of osmolality alone and osmolality + flow on mIMCD3 ET-1 mRNA levels. SIGNIFICANCE: Both flow and osmolality individually and synergistically stimulate mIMCD3 ET-1 mRNA content. These findings may be relevant to explaining high salt diet induction of CD ET-1 production.

Flow regulation of endothelin-1 production in the inner medullary collecting duct.

Collecting duct-derived endothelin (ET)-1 is an autocrine inhibitor of Na(+) and water reabsorption; its deficiency causes hypertension and water retention. Extracellular fluid volume expansion increases collecting duct ET-1, thereby promoting natriuresis and diuresis; however, how this coupling between volume expansion and collecting duct ET-1 occurs is incompletely understood. One possibility is that volume expansion increases tubular fluid flow. To investigate this, cultured IMCD3 cells were subjected to static or flow conditions. Exposure to a shear stress of 2 dyn/cm(2) for 2 h increased ET-1 mRNA content by ∼2.3-fold. Absence of perfusate Ca(2+), chelation of intracellular Ca(2+), or inhibition of Ca(2+) signaling (calmodulin, Ca(2+)/calmodulin-dependent kinase, calcineurin, PKC, or phospholipase C) prevented the flow response. Evaluation of possible flow-activated Ca(2+) entry pathways revealed no role for transient receptor potential (TRP)C3, TRPC6, and TRPV4; however, cells with TRPP2 (polycystin-2) knockdown had no ET-1 flow response. Flow increased intracellular Ca(2+) was blunted in TRPP2 knockdown cells. Nonspecific blockade of P2 receptors, as well as specific inhibition of P2X7 and P2Y2 receptors, prevented the ET-1 flow response. The ET-1 flow response was not affected by inhibition of either epithelial Na(+) channels or the mitochondrial Na(+)/Ca(2+) exchanger. Taken together, these findings provide evidence that in IMCD3 cells, flow, via polycystin-2 and P2 receptors, engages Ca(2+)-dependent signaling pathways that stimulate ET-1 synthesis.

Systemic sclerosis: current concepts in pathogenesis and therapeutic aspects of dermatological manifestations.

Systemic sclerosis (SSc) is a chronic, multisystem connective tissue disease with protean clinical manifestations. Recent advances in understanding the pathogenic mechanisms have led to development of target-oriented and vasomodulatory drugs which play a pivotal role in treating various dermatological manifestations. An exhaustive literature search was done using Medline, Embase, and Cochrane library to review the recent concepts regarding pathogenesis and evidence-based treatment of salient dermatological manifestations. The concept of shared genetic risk factors for the development of autoimmune diseases is seen in SSc. It is divided into fibroproliferative and inflammatory groups based on genome-wide molecular profiling. Genetic, infectious, and environmental factors play a key role; vascular injury, fibrosis, and immune activation are the chief pathogenic factors. Vitamin D deficiency has been documented in SSc and correlates with the severity of skin involvement. Skin sclerosis, Raynaud's phenomenon (RP) with digital vasculopathies, pigmentation, calcinosis, and leg ulcers affect the patient's quality of life. Immunosuppressives, biologicals, and hematopoietic stem cell transplantation are efficacious in skin sclerosis. Endothelin A receptor antagonists, calcium-channel blockers, angiotensin receptor inhibitors, prostacyclin analogs, and phosphodiesterase type 5 (PDE-5) inhibitors are the mainstay in RP and digital vasculopathies. Pigmentation in SSc has been attributed to melanogenic potential of endothelin-1 (ET-1); the role of ET 1 antagonists and vitamin D analogs needs to be investigated. Sexual dysfunction in both male and female patients has been attributed to vasculopathy and fibrosis, wherein PDE-5 inhibitors are found to be useful. The future concepts of treating SSc may be based on the gene expression signature.

Keratoacanthoma centrifugum marginatum at an unusual site.

Keratoacanthoma (KA) is a rapidly evolving tumor, composed of keratinizing squamous cells originating in pilosebaceous follicles and resolving spontaneously if left untreated. It is relatively uncommon in dark-skinned and occurs in middle aged individuals. Males are three times more affected than females. It presents as firm, rounded, flesh-colored or reddish papule; with a rapid growth phase followed by spontaneous healing over three months. Two types of KA exist i.e., solitary and multiple. There are three rare clinical variants of solitary KA, namely giant KA, keratoacanthoma centrifugum marginatum (KCM) and subungual KA. In KCM, lesions are large, reaching upto 20cms. There is peripheral extension with raised, rolled border and atrophy in the center. There is no tendency toward spontaneous involution. The most common locations are dorsa of hands and legs, lesions on scalp being rare. A rare case of KCM occurring on scalp which is an unusual site is reported.

Na delivery and ENaC mediate flow regulation of collecting duct endothelin-1 production.

Collecting duct (CD) endothelin-1 (ET-1) is an important autocrine inhibitor of Na and water transport. CD ET-1 production is stimulated by extracellular fluid volume expansion and tubule fluid flow, suggesting a mechanism coupling CD Na delivery and ET-1 synthesis. A mouse cortical CD cell line, mpkCCDc14, was subjected to static or flow conditions for 2 h at 2 dyn/cm(2), followed by determination of ET-1 mRNA content. Flow with 300 mosmol/l NaCl increased ET-1 mRNA to 65% above that observed under static conditions. Increasing perfusate osmolarity to 450 mosmol/l with NaCl or Na acetate increased ET-1 mRNA to ∼184% compared with no flow, which was not observed when osmolarity was increased using mannitol or urea. Reducing Na concentration to 150 mosmol/l while maintaining total osmolarity at 300 mosmol/l with urea or mannitol decreased the flow response. Inhibition of epithelial Na channel (ENaC) with amiloride or benzamil abolished the flow response, suggesting involvement of ENaC in flow-regulated ET-1 synthesis. Aldosterone almost doubled the flow response. Since Ca(2+) enhances CD ET-1 production, the involvement of plasma membrane and mitochondrial Na/Ca(2+) exchangers (NCX) was assessed. SEA0400 and KB-R7943, plasma membrane NCX inhibitors, did not affect the flow response. However, CGP37157, a mitochondrial NCX inhibitor, abolished the response. In summary, the current study indicates that increased Na delivery, leading to ENaC-mediated Na entry and mitochondrial NCX activity, is involved in flow-stimulated CD ET-1 synthesis. This constitutes the first report of either ENaC or mitochondrial NCX regulation of an autocrine factor in any biologic system.

Digital multiplexed gene expression analysis using the NanoString nCounter system.

This unit presents the protocol for the NanoString nCounter Gene Expression Assay, a robust and highly reproducible method for detecting the expression of up to 800 genes in a single reaction with high sensitivity and linearity across a broad range of expression levels. The methodology serves to bridge the gap between genome-wide (microarrays) and targeted (real-time quantitative PCR) expression profiling. The nCounter assay is based on direct digital detection of mRNA molecules of interest using target-specific, color-coded probe pairs. It does not require the conversion of mRNA to cDNA by reverse transcription or the amplification of the resulting cDNA by PCR. Each target gene of interest is detected using a pair of reporter and capture probes carrying 35- to 50-base target-specific sequences. In addition, each reporter probe carries a unique color code at the 5' end that enables the molecular barcoding of the genes of interest, while the capture probes all carry a biotin label at the 3' end that provides a molecular handle for attachment of target genes to facilitate downstream digital detection. After solution-phase hybridization between target mRNA and reporter-capture probe pairs, excess probes are removed and the probe/target complexes are aligned and immobilized in the nCounter cartridge, which is then placed in a digital analyzer for image acquisition and data processing. Hundreds of thousands of color codes designating mRNA targets of interest are directly imaged on the surface of the cartridge. The expression level of a gene is measured by counting the number of times the color-coded barcode for that gene is detected, and the barcode counts are then tabulated.