A Novel PD-L1 Antibody Promotes Antitumor Function of Peripheral Cytotoxic Lymphocytes after Radical Nephrectomy in Patients with Renal Cell Carcinoma.

The intrinsic and acquired resistance to PD-1/PD-L1 immune checkpoint blockade is an important challenge for patients and clinicians because no reliable tool has been developed to predict individualized response to immunotherapy. In this study, we demonstrate the translational relevance of an ex vivo functional assay that measures the tumor cell killing ability of patient-derived CD8 T and NK cells (referred to as "cytotoxic lymphocytes," or CLs) isolated from the peripheral blood of patients with renal cell carcinoma. Patient-derived PBMCs were isolated before and after nephrectomy from patients with renal cell carcinoma. We compared the efficacy of U.S. Food and Drug Administration (FDA)-approved PD-1/PD-L1 inhibitors (pembrolizumab, nivolumab, atezolizumab) and a newly developed PD-L1 inhibitor (H1A Ab) in eliciting cytotoxic function. CL activity was improved at 3 mo after radical nephrectomy compared with baseline, and it was associated with higher circulating levels of tumor-reactive effector CD8 T cells (CD11ahighCX3CR1+GZMB+). Treatment of PBMCs with FDA-approved PD-1/PD-L1 inhibitors enhanced tumor cell killing activity of CLs, but a differential response was observed at the individual-patient level. H1A demonstrated superior efficacy in promoting CL activity compared with FDA-approved PD-1/PD-L1 inhibitors. PBMC immunophenotyping by mass cytometry revealed enrichment of effector CD8 T and NK cells in H1A-treated PBMCs and immunosuppressive regulatory T cells in atezolizumab-treated samples. Our study lays the ground for future investigation of the therapeutic value of H1A as a next-generation immune checkpoint inhibitor and the potential of measuring CTL activity in PBMCs as a tool to predict individual response to immune checkpoint inhibitors in patients with advanced renal cell carcinoma.

Antibody-Based Therapeutics for the Treatment of Renal Cell Carcinoma: Challenges and Opportunities.

Renal cell carcinoma (RCC) is among the top 10 most common cancers in both men and women with an estimated 75 000 cases each year in the US. Over the last decade, the therapeutic landscape for patients with metastatic RCC has significantly evolved, with immunotherapy emerging as the new front-line therapy. Despite significant improvement in toxicity profile and survival outcomes, key concerns such as patient selection, treatment sequencing, and intrinsic and acquired resistance remain unresolved. Emerging options such as antibody-based therapeutics (eg, anti-CD70, anti-CA9, and anti-ENPP3) are being explored in clinical trials for patients with cancer resistant or refractory to current immunotherapies. Despite positive results for hematological cancers, breast cancer, and more recently bladder cancer, most antibody-based therapies failed to improve the outcomes in patients with advanced RCC. This underscores the need to understand the underlying causes of failed responses to this treatment class, which will ultimately support the rational design of more effective and tolerable treatments. In this review, we summarize the evolving landscape of RCC therapeutics and describe recent clinical trials with emerging antibody-based therapeutics. We also describe the challenges that need to be overcome for the successful creation of therapeutic antibodies for treating RCC.

The Impact of Metastasis Histopathology on Oncologic Outcomes for Patients With Surgically Resected Metastatic Renal Cell Carcinoma.

PURPOSE: Multiple prognostic models exist to assess survival among patients with metastatic clear cell renal cell carcinoma. However, the relative contribution of histopathological features of the metastasis has not been extensively studied. Herein, we compared models using clinical, primary tumor, and metastatic features to predict cancer-specific survival for patients with surgically resected metastatic clear cell renal cell carcinoma. MATERIALS AND METHODS: We studied 266 patients who had undergone nephrectomy between 1970 and 2019, and who had a single site of metastasis completely resected. Two versions of the metastatic clear cell renal cell carcinoma score published by Leibovich et al were calculated, using grade and necrosis from the primary tumor and using grade and necrosis from the metastasis. Predictive abilities of these 2 versions and a third model that included metastatic features only were compared using c-indexes from Cox proportional hazards models. RESULTS: A total of 197 patients died from renal cell carcinoma at a median of 2.3 years (IQR 1.1-4.5); median follow-up among survivors was 13.2 years (IQR 10.0-14.5). The Leibovich score using grade and necrosis from the metastasis (c=0.679) had similar predictive ability compared to the original Leibovich score using grade and necrosis from the primary tumor (c=0.675). A third model (c=0.707) demonstrated that metastasectomy within 2 years after nephrectomy, presence of bone metastasis, high grade, and sarcomatoid differentiation in the metastasis were significantly associated with cancer-specific survival. CONCLUSIONS: Scoring algorithms calculated using histopathological features of the metastasis can be used to predict cancer-specific survival for patients with surgically resected metastatic clear cell renal cell carcinoma. These findings are of particular importance for instances when primary tumor histopathology is not readily available.

The Interaction between the Host Genome, Epigenome, and the Gut-Skin Axis Microbiome in Atopic Dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease that occurs in genetically predisposed individuals. It involves complex interactions among the host immune system, environmental factors (such as skin barrier dysfunction), and microbial dysbiosis. Genome-wide association studies (GWAS) have identified AD risk alleles; however, the associated environmental factors remain largely unknown. Recent evidence suggests that altered microbiota composition (dysbiosis) in the skin and gut may contribute to the pathogenesis of AD. Examples of environmental factors that contribute to skin barrier dysfunction and microbial dysbiosis in AD include allergens, irritants, pollution, and microbial exposure. Studies have reported alterations in the gut microbiome structure in patients with AD compared to control subjects, characterized by increased abundance of Clostridium difficile and decreased abundance of short-chain fatty acid (SCFA)-producing bacteria such as Bifidobacterium. SCFAs play a critical role in maintaining host health, and reduced SCFA production may lead to intestinal inflammation in AD patients. The specific mechanisms through which dysbiotic bacteria and their metabolites interact with the host genome and epigenome to cause autoimmunity in AD are still unknown. By understanding the combination of environmental factors, such as gut microbiota, the genetic and epigenetic determinants that are associated with the development of autoantibodies may help unravel the pathophysiology of the disease. This review aims to elucidate the interactions between the immune system, susceptibility genes, epigenetic factors, and the gut microbiome in the development of AD.

Small RNA Profiling in an HTLV-1-Infected Patient with Acute Adult T-Cell Leukemia-Lymphoma at Diagnosis and after Maintenance Therapy: A Case Study.

Small RNAs (sRNAs) are epigenetic regulators of essential biological processes associated with the development and progression of leukemias, including adult T-cell leukemia/lymphoma (ATLL) caused by human T-cell lymphotropic virus type 1 (HTLV-1), an oncogenic human retrovirus originally discovered in a patient with adult T-cell leukemia/lymphoma. Here, we describe the sRNA profile of a 30-year-old woman with ATLL at the time of diagnosis and after maintenance therapy with the aim of correlating expression levels with response to therapy.

Applications of Plasma-Activated Water in Dentistry: A Review.

The activation of water by non-thermal plasma creates a liquid with active constituents referred to as plasma-activated water (PAW). Due to its active constituents, PAW may play an important role in different fields, such as agriculture, the food industry and healthcare. Plasma liquid technology has received attention in recent years due to its versatility and good potential, mainly focused on different health care purposes. This interest has extended to dentistry, since the use of a plasma-liquid technology could bring clinical advantages, compared to direct application of non-thermal atmospheric pressure plasmas (NTAPPs). The aim of this paper is to discuss the applicability of PAW in different areas of dentistry, according to the published literature about NTAPPs and plasma-liquid technology. The direct and indirect application of NTAPPs are presented in the introduction. Posteriorly, the main reactors for generating PAW and its active constituents with a role in biomedical applications are specified, followed by a section that discusses, in detail, the use of PAW as a tool for different oral diseases.

Effect of the pH on the Antibacterial Potential and Cytotoxicity of Different Plasma-Activated Liquids.

In this study, different plasma-activated liquids were evaluated for their antimicrobial effects against Escherichia coli, as well as for their cytotoxicity on mammalian cells. The PALs were prepared from distilled (DIS), deionized (DI), filtered (FIL), and tap (TAP) water. Additionally, 0.9% NaCl saline solution (SAL) was plasma-activated. These PALs were prepared using 5 L/min air gliding arc plasma jet for up to 60.0 min of exposure. Subsequently, the physicochemical properties, such as, the oxidation-reduction potential (ORP), the pH, the conductivity, and the total dissolved solids (TDS) were characterized by a water multiparameter. The PALs obtained showed a drastic decrease in the pH with increasing plasma exposure time, in contrast, the conductivity and TDS increased. In a general trend, the UV-vis analyses identified a higher production of the following reactive species of nitrogen and oxygen (RONS), HNO2, H2O2, NO3-, and NO2-. Except for the plasma-activated filtered water (PAW-FIL), where there was a change in the position of NO2- and NO3- at some pHs, The higher production of HNO2 and H2O2-reactive species was observed at a low pH. Finally, the standardized suspensions of Escherichia coli were exposed to PAL for up to 60.0 min. The plasma-activated deionized water (PAW-DI pH 2.5), plasma-activated distilled water (PAW-DIS pH 2.5 and 3), and plasma-activated tap water (PAW-TAP 3.5) showed the best antimicrobial effects at exposure times of 3.0, 10.0, and 30.0 min, respectively. The MTT analysis demonstrated low toxicity of all of the PAL samples. Our results indicate that the plasma activation of different liquids using the gliding arc system can generate specific physicochemical conditions that produce excellent antibacterial effects for E. coli with a safe application, thus bringing future contributions to creating new antimicrobial protocols.

The status and perspectives of nanostructured materials and fabrication processes for wearable piezoresistive sensors.

The wearable sensors have attracted a growing interest in different markets, including health, fitness, gaming, and entertainment, due to their outstanding characteristics of convenience, simplicity, accuracy, speed, and competitive price. The development of different types of wearable sensors was only possible due to advances in smart nanostructured materials with properties to detect changes in temperature, touch, pressure, movement, and humidity. Among the various sensing nanomaterials used in wearable sensors, the piezoresistive type has been extensively investigated and their potential have been demonstrated for different applications. In this review article, the current status and challenges of nanomaterials and fabrication processes for wearable piezoresistive sensors are presented in three parts. The first part focuses on the different types of sensing nanomaterials, namely, zero-dimensional (0D), one-dimensional (1D), two-dimensional (2D), and three-dimensional (3D) piezoresistive nanomaterials. Then, in second part, their fabrication processes and integration are discussed. Finally, the last part presents examples of wearable piezoresistive sensors and their applications.

Preconceptional Immunization Can Modulate Offspring Intrathymic IL-17-Producing γδT Cells with Epigenetic Implications Mediated by microRNAs.

The mechanisms through which maternal immunization can modulate offspring thymic maturation of lymphocytes are not fully understood. Here, we aimed to evaluate whether maternal OVA-immunization can inhibit the maturation of IL-17-producing γδT cells in offspring thymus, and if this mechanism has epigenetic implications mediated by microRNAs (miRNAs) expression. Wild-type (WT) C57BL/6 females were immunized with OVA in Alum or Alum alone and were mated with normal WT males. Evaluating their offspring thymus at 3 or 20 days old (d.o.), we observed that maternal OVA immunization could inhibit the thymic frequency of offspring CD27- and IL-17+ γδT cells at the neonatal and until 20 days old. Furthermore, we evaluated the expression of function-related γ and δ variable γδTCR chains (Vγ1, Vγ2, Vγ3, Vδ4, and Vδ6.3), observing that maternal OVA-immunization inhibits Vγ2 chains expression. The small RNAs (sRNAs), particularly miRNAs, and messenger RNAs (mRNA) expression profiles by pools of thymus tissue samples (from 9 to 11 mice) from offspring OVA-immunized or Alum-immunized mothers were analyzed via Illumina sequencing platform and bioinformatics approaches. Using a fold change >4, our results showed that seven miRNAs (mmu-miR-126a-3p, 101a-3p, 744-3p,142-5p, 15a-5p, 532-5p, and 98-5p) were differentially expressed between both groups. Ten target genes were predicted to interact with the seven selected miRNAs. There were no enriched categories of gene ontology functional annotation and pathway enrichment analysis for the target genes. Interestingly, four of the identified miRNAs (mmu-miR-15a, mmu-miR-101 mmu-miR-126, and mmu-miR-142) are related to IL-17 production. Our data is of significance because we demonstrate that maternal immunization can modulate offspring thymic maturation of IL-17-producing γδT cells possibly by an epigenetic mechanism mediated by miRNAs.

Rabies virus diversification in aerial and terrestrial mammals.

Rabies is a fatal zoonotic infection of the central nervous system of mammals and has been known to humans for millennia. The etiological agent, is a neurotropic RNA virus in the order Mononegavirales, family Rhabdoviridae, genus Lyssavirus. There are currently accepted to be two cycles for rabies transmission: the urban cycle and the sylvatic cycle. The fact that both cycles originated from a common RABV or lyssavirus ancestor and the adaptive divergence that occurred since then as this ancestor virus adapted to a wide range of fitness landscapes represented by reservoir species in the orders Carnivora and Chiroptera led to the emergence of the diverse RABV lineages currently found in the sylvatic and urban cycles. Here we study full genome phylogenies and the time to the most recent common ancestor (TMRCA) of the RABVs in the sylvatic and urban cycles. Results show that there were differences between the nucleotide substitution rates per site per year for the same RABV genes maintained independently in the urban and sylvatic cycles. The results identify the most suitable gene for phylogenetic analysis, heterotachy among RABV genes and the TMRCA for the two cycles.

Value of 3-Tesla multiparametric magnetic resonance imaging and targeted biopsy for improved risk stratification in patients considered for active surveillance.

OBJECTIVE: To evaluate the role of multiparametric magnetic resonance imaging (mpMRI) of the prostate and transrectal ultrasonography guided biopsy (TRUS-Bx) with visual estimation in early risk stratification of patients with prostate cancer on active surveillance (AS). PATIENTS AND METHODS: Patients with low-risk, low-grade, localised prostate cancer were prospectively enrolled and submitted to a 3-T 16-channel cardiac surface coil mpMRI of the prostate and confirmatory biopsy (CBx), which included a standard biopsy (SBx) and visual estimation-guided TRUS-Bx. Cancer-suspicious regions were defined using Prostate Imaging Reporting and Data System (PI-RADS) scores. Reclassification occurred if CBx confirmed the presence of a Gleason score ≥7, greater than three positive fragments, or ≥50% involvement of any core. The performance of mpMRI for the prediction of CBx results was assessed. Univariate and multivariate logistic regressions were performed to study relationships between age, prostate-specific antigen (PSA) level, PSA density (PSAD), number of positive cores in the initial biopsy, and mpMRI grade on CBx reclassification. Our report is consistent with the Standards of Reporting for MRI-targeted Biopsy Studies (START) guidelines. RESULTS: In all, 105 patients were available for analysis in the study. From this cohort, 42 (40%) had PI-RADS 1, 2, or 3 lesions and 63 (60%) had only grade 4 or 5 lesions. Overall, 87 patients underwent visual estimation TRUS-Bx. Reclassification among patients with PI-RADS 1, 2, 3, 4, and 5 was 0%, 23.1%, 9.1%, 74.5%, and 100%, respectively. Overall, mpMRI sensitivity, specificity, positive predictive value, and negative predictive value for disease reclassification were 92.5%, 76%, 81%, and 90.5%, respectively. In the multivariate analysis, only PSAD and mpMRI remained significant for reclassification (P < 0.05). In the cross-tabulation, SBx would have missed 15 significant cases detected by targeted biopsy, but SBx did detect five cases of significant cancer not detected by targeted biopsy alone. CONCLUSION: Multiparametric magnetic resonance imaging is a significant tool for predicting cancer severity reclassification on CBx among AS candidates. The reclassification rate on CBx is particularly high in the group of patients who have PI-RADS grades 4 or 5 lesions. Despite the usefulness of visual-guided biopsy, it still remains highly recommended to retrieve standard fragments during CBx in order to avoid missing significant tumours.

A prognostic model for survival after palliative urinary diversion for malignant ureteric obstruction: a prospective study of 208 patients.

OBJECTIVE: To identify factors associated with survival after palliative urinary diversion (UD) for patients with malignant ureteric obstruction (MUO) and create a risk-stratification model for treatment decisions. PATIENTS AND METHODS: We prospectively collected clinical and laboratory data for patients who underwent palliative UD by ureteric stenting or percutaneous nephrostomy (PCN) between 1 January 2009 and 1 November 2011 in two tertiary care university hospitals, with a minimum 6-month follow-up. Inclusion criteria were age >18 years and MUO confirmed by computed tomography, ultrasonography or magnetic resonance imaging. Factors related to poor prognosis were identified by Cox univariable and multivariable regression analyses, and a risk stratification model was created by Kaplan-Meier survival estimates at 1, 6 and 12 months, and log-rank tests. RESULTS: The median (range) survival was 144 (0-1084) days for the 208 patients included after UD (58 ureteric stenting, 150 PCN); 164 patients died, 44 (21.2%) during hospitalisation. Overall survival did not differ by UD type (P = 0.216). The number of events related to malignancy (≥4) and Eastern Cooperative Oncology Group (ECOG) index (≥2) were associated with short survival on multivariable analysis. These two risk factors were used to divide patients into three groups by survival type: favourable (no factors), intermediate (one factor) and unfavourable (two factors). The median survival at 1, 6, and 12 months was 94.4%, 57.3% and 44.9% in the favourable group; 78.0%, 36.3%, and 15.5% in the intermediate group; and 46.4%, 14.3%, and 7.1% in the unfavourable group (P < 0.001). CONCLUSIONS: Our stratification model may be useful to determine whether UD is indicated for patients with MUO.

Detection of Zika virus in Brazilian patients during the first five days of infection - urine versus plasma.

Advantages of testing for Zika virus (ZIKV) in urine have been reported, such as the persistence of ZIKV in this type of specimen for up to 20 days after ZIKV disease onset. We investigate 61 patients in the first 5 days post-symptom onset and find more patients testing positive for ZIKV in plasma samples (n=46), than in corresponding urine samples (n=37). For patients respectively testing positive in both plasma and urine (n=28), respective viral loads appeared similar.

Mitochondrial genomes and comparative analyses of Culex camposi, Culex coronator, Culex usquatus and Culex usquatissimus (Diptera:Culicidae), members of the coronator group.

BACKGROUND: The Coronator Group currently encompasses six morphologically similar species (Culex camposi Dyar, Culex coronator Dyar and Knab, Culex covagarciai Forattini, Culex usquatus Dyar, Culex usquatissimus Dyar, and Culex ousqua Dyar). Culex coronator has been incriminated as a potential vector of West Nile Virus (WNV), Saint Louis Encephalitis Virus (SLEV), and Venezuelan Equine Encephalitis Virus (VEEV). The complete mitochondrial genome of Cx. coronator, Cx. usquatus, Cx.usquatissimus, and Cx. camposi was sequenced, annotated, and analyzed to provide genetic information about these species. RESULTS: The mitochondrial genomes of Cx. coronator, Cx. usquatus, Cx.usquatissimus, and Cx. camposi varied from 15,573 base pairs in Cx. usquatus to 15,576 in Cx. coronator. They contained 37 genes (13 protein-encoding genes, 2 rRNA genes, and 22 tRNA genes) and the AT-rich control region. Comparative analyses of the 37 genes demonstrated the mitochondrial genomes to be composed of variable and conserved genes. Despite the small size, the ATP8, ATP6 plus NADH5 protein-encoding genes were polymorphic, whereas tRNAs and rRNAs were conserved. The control region contained some poly-T stretch. The Bayesian phylogenetic tree corroborated that both the Coronator Group and the Culex pipens complex are monophyletic taxa. CONCLUSIONS: The mitochondrial genomes of Cx. coronator, Cx. usquatus, Cx. usquatissimus and Cx. camposi share the same gene composition and arrangement features that match to those reported for most Culicidae species. They are composed of the same 37 genes and the AT-rich control region, which contains poly-T stretches that may be involved in the functional role of the mitochondrial genome. Taken together, results of the dN/dS ratios, the sliding window analyses and the Bayesian phylogenetic analyses suggest that ATP6, ATP8 and NADH5 are promising genes to be employed in phylogenetic studies involving species of the Coronator Group, and probably other species groups of the subgenus Culex. Bayesian topology corroborated the morphological hypothesis of the Coronator Group as monophyletic lineage within the subgenus Culex.

Enhanced detection of viral diversity using partial and near full-length genomes of human immunodeficiency virus Type 1 provirus deep sequencing data from recently infected donors at four blood centers in Brazil.

BACKGROUND: Here, we report application of high-throughput near full-length genome (NFLG) and partial human immunodeficiency virus Type 1 (HIV-1) proviral genome deep sequencing to characterize HIV in recently infected blood donors at four major blood centers in Brazil. STUDY DESIGN AND METHODS: From 2007 to 2011, a total of 341 HIV+ blood donors from four blood centers were recruited to participate in a case-control study to identify HIV risk factors and motivations to donate. Forty-seven (17 from São Paulo, eight from Minas Gerais, 11 from Pernambuco, and 11 from Rio de Janeiro) were classified as recently infected based on testing by less-sensitive enzyme immunoassays. Five overlapping amplicons spanning the HIV genome were polymerase chain reaction amplified from peripheral blood mononuclear cells. The amplicons were molecularly barcoded, pooled, and sequenced by a paired-end protocol (Illumina). RESULTS: Of the 47 recently infected donor samples studied, 39 (82.9%) NFLGs and six (12.7%) partial fragments were de novo assembled into contiguous sequences and successfully subtyped. Subtype B was the only nonrecombinant virus identified in this study and accounted for 62.2% (28/45) of samples. The remaining 37.8% (17/45) of samples showed various patterns of subtype discordance in different regions of HIV-1 genomes, indicating two to four circulating recombinant subtypes derived from Clades B, F, and C. Fourteen samples (31.1%) from this study harbored drug resistance mutations, indicating higher rate of drug resistance among Brazilian blood donors. CONCLUSION: Our findings revealed a high proportion of HIV-1 recombinants among recently infected blood donors in Brazil, which has implications for future blood screening, diagnosis, therapy, and vaccine development.

Frequency of coreceptor tropism in PBMC samples from HIV-1 recently infected blood donors by massively parallel sequencing: the REDS II study.

BACKGROUND: The interaction of HIV-1 and target cells involves sequential binding of the viral gp120 Env protein to the CD4 receptor and a chemokine co-receptor (either CCR5 or CXCR4). CCR5 antagonists have proved to be an effective salvage therapy in patients with CCR5 using variants (R5) but not with variants capable of using CXCR4 (×4) phenotype. Thus, it is critically important to determine cellular tropism of a country's circulating HIV strains to guide a management decision to improve treatment outcome. In this study, we report the prevalence of R5 and ×4 HIV strains in 45 proviral DNA massively parallel sequencing "MPS" data from recently infected Brazilian blood donors. METHODS: The MPS data encompassing the tropism-related V3 loop region of the HIV-1 env gene was extracted from our recently published HIV-1 genomes sequenced by a paired-end protocol (Illumina). HIV-1 tropism was inferred using Geno2pheno[coreceptor] algorithm (3.5 % false-positive rate). V3 net charge and 11/25 rules were also used for coreceptor prediction. RESULTS: Among the 45 samples for which tropism were determined, 39 were exclusively R5 variants, 5 ×4 variants, and one dual-tropic or mixed (D/M) populations of R5 and ×4 viruses, corresponding to 86.7, 11.1 and 2.2 %, respectively. Thus, the proportion of all blood donors that harbor CXCR4-using virus was 13.3 % including individuals with D/M-tropic viruses. CONCLUSIONS: The presence of CCR5-tropic variants in more than 85 % of our cohort of antiretroviral-naïve blood donors with recent HIV-1 infection indicates a potential benefit of CCR5 antagonists as a therapeutic option in Brazil. Therefore, determination of viral co-receptor tropism is an important diagnostic prerequisite.

Phylogeny and historical biogeography of gnateaters (Passeriformes, Conopophagidae) in the South America forests.

We inferred the phylogenetic relationships, divergence time and biogeography of Conopophagidae (gnateaters) based on sequence data of mitochondrial genes (ND2, ND3 and cytb) and nuclear introns (TGFB2 and G3PDH) from 45 tissue samples (43 Conopophaga and 2 Pittasoma) representing all currently recognized species of the family and the majority of subspecies. Phylogenetic relationships were estimated by maximum likelihood and Bayesian inference. Divergence time estimates were obtained based on a Bayesian relaxed clock model. These chronograms were used to calculate diversification rates and reconstruct ancestral areas of the genus Conopophaga. The phylogenetic analyses support the reciprocal monophyly of the two genera, Conopophaga and Pittasoma. All species were monophyletic with the exception of C. lineata, as C. lineata cearae did not cluster with the other two C. lineata subspecies. Divergence time estimates for Conopophagidae suggested that diversification took place during the Neogene, and that the diversification rate within Conopophaga clade was highest in the late Miocene, followed by a slower diversification rate, suggesting a diversity-dependent pattern. Our analyses of the diversification of family Conopophagidae provided a scenario for evolution in Terra Firme forest across tropical South America. The spatio-temporal pattern suggests that Conopophaga originated in the Brazilian Shield and that a complex sequence of events possibly related to the Andean uplift and infilling of former sedimentation basins and erosion cycles shaped the current distribution and diversity of this genus.