Peno-scrotal metastasis of colorectal adenocarcinoma: A case report and review of the literature.

INTRODUCTION: Colorectal cancer is the second most common cause of death worldwide and may present as metastatic disease involving the liver, lungs and bones, and pelvic organs. Penile and scrotal metastasis may occur secondary to primary tumors originating from the genito-urinary tract. CASE PRESENTATION: We present a case of rectal adenocarcinoma metastatic to the penis and scrotum s/p chemotherapy and radiotherapy. His metastatic involvement was complicated by infections, necrosis and growth of maggots. Patient underwent wide excision and debridement of the metastatic deposits in the pubic, penile, scrotal and perineal region in addition to split-tissue skin graft from the right anterior thigh to cover the pubic region, the remainder of the penile shaft, and the scrotum. CLINICAL DISCUSSION: Peno-scrotal metastasis secondary to rectal adenocarcinoma is a rare condition. It may present as malignant priapism, hematuria, obstructive urinary symptoms occurring when there is urethral involvement. The mechanism of metastasis may be due to retrograde venous flow from the vesical, pudendal, hemorrhoidal plexuses, and the prostatic venous plexus draining the cavernosal veins of the penis. Additionally, lymphatic involvement and spread may play an important role in the spread of the disease. Treatment of such conditions relies on chemotherapy and radiotherapy and surgery for local control and symptomatic treatment. CONCLUSION: This article describes the rare occurrence of peno-scrotal metastasis of rectal adenocarcinoma and provides an overview on diagnosis, etiology, and management of the disease.

Deep mutational scanning of Pneumocystis jirovecii dihydrofolate reductase reveals allosteric mechanism of resistance to an antifolate.

Pneumocystis jirovecii is a fungal pathogen that causes pneumocystis pneumonia, a disease that mainly affects immunocompromised individuals. This fungus has historically been hard to study because of our inability to grow it in vitro. One of the main drug targets in P. jirovecii is its dihydrofolate reductase (PjDHFR). Here, by using functional complementation of the baker's yeast ortholog, we show that PjDHFR can be inhibited by the antifolate methotrexate in a dose-dependent manner. Using deep mutational scanning of PjDHFR, we identify mutations conferring resistance to methotrexate. Thirty-one sites spanning the protein have at least one mutation that leads to resistance, for a total of 355 high-confidence resistance mutations. Most resistance-inducing mutations are found inside the active site, and many are structurally equivalent to mutations known to lead to resistance to different antifolates in other organisms. Some sites show specific resistance mutations, where only a single substitution confers resistance, whereas others are more permissive, as several substitutions at these sites confer resistance. Surprisingly, one of the permissive sites (F199) is without direct contact to either ligand or cofactor, suggesting that it acts through an allosteric mechanism. Modeling changes in binding energy between F199 mutants and drug shows that most mutations destabilize interactions between the protein and the drug. This evidence points towards a more important role of this position in resistance than previously estimated and highlights potential unknown allosteric mechanisms of resistance to antifolate in DHFRs. Our results offer unprecedented resources for the interpretation of mutation effects in the main drug target of an uncultivable fungal pathogen.

HIV risk behaviour, viraemia, and transmission across HIV cascade stages including low-level viremia: Analysis of 14 cross-sectional population-based HIV Impact Assessment surveys in sub-Saharan Africa.

As antiretroviral treatment (ART) coverage for people living with HIV (PLHIV) increases, HIV programmes require up-to-date information about evolving HIV risk behaviour and transmission risk, including those with low-level viremia (LLV; >50 to ≤1000 copies/mL), to guide prevention priorities. We aimed to assess differences in sexual risk behaviours, distribution of viral load (VL) and proportion of transmission across PLHIV subgroups. We analysed data from Population-based HIV Impact Assessment surveys in 14 sub-Saharan African countries during 2015-2019. We estimated adjusted prevalence ratios (aPR) of self-reported HIV high-risk behaviour (multiple partners and condomless sex) across cascade stages via generalised estimation equations. We modelled the proportions of transmission from each subgroup using relative self-reported sexual risk, a Hill function for transmission rate by VL, and proportions within cascade stages from surveys and UNAIDS country estimates for 2010-2020. Compared to PLHIV with undetectable VL (≤50 copies/mL), undiagnosed PLHIV (aPR women: 1.28 [95% CI: 1.08-1.52]; men: 1.61 [1.33-1.95]) and men diagnosed but untreated (2.06 [1.52-2.78]) were more likely to self-report high-risk sex. High-risk behaviour was not significantly associated with LLV. Mean VL was similar among undiagnosed, diagnosed but untreated, and on ART but non-suppressed sub-groups. Across surveys, undiagnosed and diagnosed but untreated contributed most to transmission (40-91% and 1-41%, respectively), with less than 1% from those with LLV. Between 2010 and 2020, the proportion of transmission from individuals on ART but non-suppressed increased. In settings with high ART coverage, effective HIV testing, ART linkage, and retention remain priorities to reduce HIV transmission. Persons with LLV are an increasing share of PLHIV but their contribution to HIV transmission was small. Improving suppression among PLHIV on ART with VL ≥1000 copies/mL will become increasingly important.

Quantitative Peptidomics Using Reductive Methylation of Amines.

A number of different approaches have been used for quantitative peptidomics. In this protocol, we describe the method in which peptides are reacted with formaldehyde and sodium cyanoborohydride, which converts primary and secondary amines into tertiary amines. By using different combinations of regular reagents, deuterated reagents (2H), and reagents containing deuterium and 13C, it is possible to produce five isotopically distinct forms of the methylated peptides, which can be quantified by mass spectrometry. Peptides with free N-termini that are primary amines incorporate two methyl groups using this procedure, which differ by 2 Da for each of the five isotopic combinations. Peptides that contain unmodified lysine residues incorporate additional pairs of methyl groups, leading to larger mass differences between isotopic forms. The reagents are commercially available, relatively inexpensive, and chemically stable.

Analysis of the Snake Venom Peptidome.

Snake venom peptidomes are known to be a large source of molecules with different pharmacological properties. The complexity and variability of snake venoms, the presence of proteinases, and the lack of complete species-specific genome sequences make snake venom peptidome profiling a challenging task that requires especial technical strategies for sample processing and mass spectrometric analysis. Here, we describe a method for assessing the content of snake venom peptides and highlight the importance of sampling procedures, as they substantially influence the peptidomic complexity of snake venoms.

Identification of Peptides in Spider Venom Using Mass Spectrometry.

Spider venoms are composed of hundreds of proteins and peptides. Several of these venom toxins are cysteine-rich peptides in the mass range of 3-9 kDa. Small peptides (<3 kDa) can be fully characterized by mass spectrometry analysis, while proteins are generally identified by the bottom-up approach in which proteins are first digested with trypsin to generate shorter peptides for MS/MS characterization. In general, it is sufficient for protein identification to sequence two or more peptides, but for venom peptidomics it is desirable to completely elucidate peptide sequences and the number of disulfide bonds in the molecules. In this chapter, we describe a methodology to completely sequence and determine the number of disulfide bonds of spider venom peptides in the mass range of 3-9 kDa by multiple enzyme digestion, mass spectrometry of native and digested peptides, de novo analysis, and sequence overlap alignment.

Cryptic genetic variation shapes the fate of gene duplicates in a protein interaction network.

Paralogous genes are often redundant for long periods of time before they diverge in function. While their functions are preserved, paralogous proteins can accumulate mutations that, through epistasis, could impact their fate in the future. By quantifying the impact of all single-amino acid substitutions on the binding of two myosin proteins to their interaction partners, we find that the future evolution of these proteins is highly contingent on their regulatory divergence and the mutations that have silently accumulated in their protein binding domains. Differences in the promoter strength of the two paralogs amplify the impact of mutations on binding in the lowly expressed one. While some mutations would be sufficient to non-functionalize one paralog, they would have minimal impact on the other. Our results reveal how functionally equivalent protein domains could be destined to specific fates by regulatory and cryptic coding sequence changes that currently have little to no functional impact.

A new R package to parse plant species occurrence records into unique collection events efficiently reduces data redundancy.

Biodiversity data aggregators, such as Global Biodiversity Information Facility (GBIF) suffer from inflation of the number of occurrence records when data from different databases are merged but not fully reconciled. The ParseGBIF workflow is designed to parse duplicate GBIF species occurrence records into unique collection events (gatherings) and to optimise the quality of the spatial data associated with them. ParseGBIF provides tools to verify and standardize species scientific names according to the World Checklist of Vascular Plants taxonomic backbone, and to parse duplicate records into unique 'collection events', in the process compiling the most informative spatial data, where more than one duplicate is available, and providing crude estimates of taxonomic and spatial data quality. When GBIF occurrence records for a medium-sized vascular plant family, the Myrtaceae, were processed by ParseGBIF, the average number of records useful for spatial analysis increased by 180%. ParseGBIF could therefore be valuable in the evaluation of species' occurrences at the national scale in support for national biodiversity plans, identification of plant areas important for biodiversity, sample bias estimation to inform future sampling efforts, and to forecast species range shifts in response to global climate change.

Compensatory mutations potentiate constructive neutral evolution by gene duplication.

Protein functions generally depend on their assembly into complexes. During evolution, some complexes have transitioned from homomers encoded by a single gene to heteromers encoded by duplicate genes. This transition could occur without adaptive evolution through intermolecular compensatory mutations. Here, we experimentally duplicate and evolve an homodimeric enzyme to examine if and how this could happen. We identify hundreds of deleterious mutations that inactivate individual homodimers but produce functional enzymes when co-expressed as duplicated proteins that heterodimerize. The structure of one such heteromer reveals how both losses of function are buffered through the introduction of asymmetry in the complex that allows them to subfunctionalize. Constructive neutral evolution can thus occur by gene duplication followed by only one deleterious mutation per duplicate.

Dissection of the role of a Src homology 3 domain in the evolution of binding preference of paralogous proteins.

Protein-protein interactions (PPIs) drive many cellular processes. Some interactions are directed by Src homology 3 (SH3) domains that bind proline-rich motifs on other proteins. The evolution of the binding specificity of SH3 domains is not completely understood, particularly following gene duplication. Paralogous genes accumulate mutations that can modify protein functions and, for SH3 domains, their binding preferences. Here, we examined how the binding of the SH3 domains of 2 paralogous yeast type I myosins, Myo3 and Myo5, evolved following duplication. We found that the paralogs have subtly different SH3-dependent interaction profiles. However, by swapping SH3 domains between the paralogs and characterizing the SH3 domains freed from their protein context, we find that very few of the differences in interactions, if any, depend on the SH3 domains themselves. We used ancestral sequence reconstruction to resurrect the preduplication SH3 domains and examined, moving back in time, how the binding preference changed. Although the most recent ancestor of the 2 domains had a very similar binding preference as the extant ones, older ancestral domains displayed a gradual loss of interaction with the modern interaction partners when inserted in the extant paralogs. Molecular docking and experimental characterization of the free ancestral domains showed that their affinity with the proline motifs is likely not the cause for this loss of binding. Taken together, our results suggest that a SH3 and its host protein could create intramolecular or allosteric interactions essential for the SH3-dependent PPIs, making domains not functionally equivalent even when they have the same binding specificity.

An integrative approach to species delimitation sinks three Chinese limestone karst Elatostema (Urticaceae) species.

Elatostema is recognized as a taxonomically difficult group due to the reduced nature of the tiny flowers and inflorescences, also the large number of species (ca 650 to 700). Different opinions on morphological species delimitation have resulted in instability, which is problematic in such a speciose group. In this paper, the taxonomic status of three putative species, E.robustipes, E.scaposum, E.conduplicatum and their hypothetical closest relatives, was revised using morphological and molecular observations. Morphological comparison suggested high similarity between E.robustipes & E.retrohirtum, E.scaposum & E.oblongifolium, E.conduplicatum & E.coriaceifolium, respectively. Phylogenetic analyses of four universal DNA barcodes (ITS, trnH-psbA, matK and rbcL) suggested that each species pair represents a single evolutionary lineage. Taking these two findings together, we propose E.robustipes to be a synonym of E.retrohirtum, E.scaposum a synonym of E.oblongifolium, and E.conduplicatum a synonym of E.coriaceifolium. Our results recover the number, shape and size of the bracts and bracteoles to be relatively stable characters, and the disposition of the male inflorescences on modified stems to be an unstable character, unsuitable for species delimitation in Elatostema.

An interdisciplinary therapy for lifestyle change is effective in improving psychological and inflammatory parameters in women with grade I obesity.

Obesity and depression, disorders associated with inflammation, have high incidences in women. Understanding the derangements present in the initial phase of obesity may point to factors that could help avoiding disease aggravation. The present study aimed at investigating the effects of a 6-months interdisciplinary therapy for weight loss in women with grade I obesity. Before and after the therapy, 37 middle-aged women donated blood and responded to questionnaires for depression and anxiety symptoms. Inflammatory parameters were evaluated in serum and a preliminary screening of the plasma proteome was performed. The therapy decreased anthropometric, psychological scores, and serum levels of inflammatory parameters. Depression and anxiety scores correlated positively with some inflammatory parameters. The proteomic analysis showed changes in proteins related to cholesterol metabolism and inflammatory response. Interdisciplinary therapy improves anthropometric and inflammatory statuses and ameliorating psychological symptoms. The decrease of MCP-1 levels after interdisciplinary therapy has not been reported so far, at the best of our knowledge. The present demonstration of positive associations of inflammatory markers and psychological scores indicate that these mediators may be useful to monitor psychological status in obesity. The present proteome data, although preliminary, pointed to plasma alterations indicative of improvement of inflammation after interdisciplinary therapy.

Parallel Nonfunctionalization of CK1δ/ε Kinase Ohnologs Following a Whole-Genome Duplication Event.

Whole-genome duplication (WGD) followed by speciation allows us to examine the parallel evolution of ohnolog pairs. In the yeast family Saccharomycetaceae, HRR25 is a rare case of repeated ohnolog maintenance. This gene has reverted to a single copy in Saccharomyces cerevisiae where it is now essential, but has been maintained as pairs in at least 7 species post-WGD. In S. cerevisiae, HRR25 encodes the casein kinase 1δ/ε and plays a role in a variety of functions through its kinase activity and protein-protein interactions (PPIs). We hypothesized that the maintenance of duplicated HRR25 ohnologs could be a result of repeated subfunctionalization. We tested this hypothesis through a functional complementation assay in S. cerevisiae, testing all pairwise combinations of 25 orthologs (including 7 ohnolog pairs). Contrary to our expectations, we observed no cases of pair-dependent complementation, which would have supported the subfunctionalization hypothesis. Instead, most post-WGD species have one ohnolog that failed to complement, suggesting their nonfunctionalization or neofunctionalization. The ohnologs incapable of complementation have undergone more rapid protein evolution, lost most PPIs that were observed for their functional counterparts and singletons from post-WGD and non-WGD species, and have nonconserved cellular localization, consistent with their ongoing loss of function. The analysis in Naumovozyma castellii shows that the noncomplementing ohnolog is expressed at a lower level and has become nonessential. Taken together, our results indicate that HRR25 orthologs are undergoing gradual nonfunctionalization.

How diverse are the mountain karst forests of Mexico?

Tropical forests on karstic relief (tropical karst forest) are among the most species-rich biomes. These forests play pivotal roles as global climate regulators and for human wellbeing. Their long-term conservation could be central to global climate mitigation and biodiversity conservation. In Mexico, karst landscapes occupy 20% of the total land surface and are distributed mainly in the southeast of the country, along the eastern slope, and in the Yucatan Peninsula. Within each of these areas, the following types of karst occur: coastal karst, plain karst, hill karst, and mountain karst (low, medium, high). Mountain karst cover 2.07% of Mexico's land surface and are covered by tropical rainforests, montane cloud forests, and tropical deciduous forests. These are probably one of the most diverse biomes in Mexico. However, the mountain karst forests of Mexico have received little attention, and very little is known about their diversity. Here, we evaluated the vascular plant species richness within the mountain karst forests of Mexico. We assembled the first, largest, and most comprehensive datasets of Mexican mountain karst forest species, from different public databases (CONABIO, GBIF, IBdata-UNAM), which included a critical review of all data. We compiled a list of the families, genera, and species present within the mountain karst forests of Mexico. Taxa that best characterize these forests were identified based on their spatial correlation with this biome. We explored biodiversity patterns, identifying areas with the highest species richness, endemism centers, and areas of relatively low sampling intensity. We found that within the mountain karst forests of Mexico there are representatives of 11,771 vascular plant species (253 families and 2,254 genera), ca. 50% of the Mexican flora. We identified 372 species endemic to these forests. According to preliminary IUCN red list criteria, 2,477 species are under some category of conservation risk, of which 456 (3.8%) are endangered. Most of the Mexican mountain karst forests have been extensively explored and six allopatric, species-rich areas were identified. Compared to other regions in the world, the mountain karst forests of Mexico are one of the most diverse biomes. They contain more species than some entire montane systems in Mexico such as Sierra Madre Oriental, and Sierra Madre del Sur. Also, the mountain karst forests of Mexico are most diverse than similar forests of South America and Asia, even if considering the effect of different sampling areas. The fact that mountain karst forests are embedded in areas of high biotic diversity, probably contributes to their great floristic diversity. Thus, the mountain karst forests of Mexico are an important source of diversity and shelters a large percentage of the Mexican flora.

Hepatitis C virus prevalence among men who have sex with men: a cross-sectional study in 12 Brazilian cities.

BACKGROUND: Despite the preventive policies adopted, reduction in sexually transmitted infections (STIs) among men who have sex with men (MSM) has been limited. The risk of hepatitis C virus (HCV) infection has increased among the most vulnerable population groups, including MSM. The aim of this study was to estimate the prevalence of HCV infection and to assess risky practices among MSM from 12 Brazilian cities. METHODS: This study was carried out from June to December 2016 using respondent driven sampling (RDS). Participants completed a self-administered questionnaire to collect behavioral, socioeconomic, and demographic variables. In addition, the rapid diagnostic test (RDT) for HCV was offered. Positive results were sent to Instituto Adolfo Lutz for confirmation. RESULTS: A total of 4,176 participants were recruited and 23 samples were sent for confirmation. Of these, 16 were confirmed, resulting in a prevalence of 0.7% (95% CI: 0.3%-1.7%). The Southeast region showed a prevalence of 0.9% (95% CI: 0.3-2.6), followed by the South region, with 0.6% (95% CI: 0.2-2.1). The Northeast region had a prevalence of 0.3% (95% CI: 0.1-1.0) and the Midwest 0.1% (95% CI: 0.0-0.7). No positive cases were found in the North. Single men aged 40 years or older were the majority of participants exposed to HCV. High levels of alcohol consumption, illicit drug use, irregular condom use, in addition to infection with other STIs, were associated with exposure to HCV. CONCLUSIONS: STIs continue to be important health problems in Brazil and globally. Many STIs are inapparent for many years until they bring more serious consequences. Extra investment in HCV is also warranted, given that it can be eliminated. Relying solely on clinical data to provide information about inapparent infection, especially in stigmatized populations, will make that goal more difficult to achieve. Surveillance studies, such as the one reported here need to be repeated over time to demonstrate trends and to provide information for evaluation, program and policies. Investments in the most vulnerable populations are critical to achieve the World Health Organization global health goals including the elimination of viral hepatitis by 2030.

Cross-feeding affects the target of resistance evolution to an antifungal drug.

Pathogenic fungi are a cause of growing concern. Developing an efficient and safe antifungal is challenging because of the similar biological properties of fungal and host cells. Consequently, there is an urgent need to better understand the mechanisms underlying antifungal resistance to prolong the efficacy of current molecules. A major step in this direction would be to be able to predict or even prevent the acquisition of resistance. We leverage the power of experimental evolution to quantify the diversity of paths to resistance to the antifungal 5-fluorocytosine (5-FC), commercially known as flucytosine. We generated hundreds of independent 5-FC resistant mutants derived from two genetic backgrounds from wild isolates of Saccharomyces cerevisiae. Through automated pin-spotting, whole-genome and amplicon sequencing, we identified the most likely causes of resistance for most strains. Approximately a third of all resistant mutants evolved resistance through a pleiotropic drug response, a potentially novel mechanism in response to 5-FC, marked by cross-resistance to fluconazole. These cross-resistant mutants are characterized by a loss of respiration and a strong tradeoff in drug-free media. For the majority of the remaining two thirds, resistance was acquired through loss-of-function mutations in FUR1, which encodes an important enzyme in the metabolism of 5-FC. We describe conditions in which mutations affecting this particular step of the metabolic pathway are favored over known resistance mutations affecting a step upstream, such as the well-known target cytosine deaminase encoded by FCY1. This observation suggests that ecological interactions may dictate the identity of resistance hotspots.

Parallel nonfunctionalization of CK1δ/ε kinase ohnologs following a whole-genome duplication event.

Whole genome duplication (WGD) followed by speciation allows us to examine the parallel evolution of ohnolog pairs. In the yeast family Saccharomycetaceae, HRR25 is a rare case of repeated ohnolog maintenance. This gene has reverted to a single copy in S. cerevisiae where it is now essential, but has been maintained as pairs in at least 7 species post WGD. In S. cerevisiae, HRR25 encodes the casein kinase (CK) 1δ/ε and plays a role in a variety of functions through its kinase activity and protein-protein interactions (PPIs). We hypothesized that the maintenance of duplicated HRR25 ohnologs could be a result of repeated subfunctionalization. We tested this hypothesis through a functional complementation assay in S. cerevisiae, testing all pairwise combinations of 25 orthologs (including 7 ohnolog pairs). Contrary to our expectations, we observed no cases of pair-dependent complementation, which would have supported the subfunctionalization hypothesis. Instead, most post-WGD species have one ohnolog that failed to complement, suggesting their nonfunctionalization or neofunctionalization. The ohnologs incapable of complementation have undergone more rapid protein evolution, lost most PPIs that were observed for their functional counterparts and singletons from post and non-WGD species, and have non-conserved cellular localization, consistent with their ongoing loss of function. The analysis in N. castelli shows that the non-complementing ohnolog is expressed at a lower level and has become non-essential. Taken together, our results indicate that HRR25 orthologs are undergoing gradual nonfunctionalization.

The fitness cost of spurious phosphorylation.

The fidelity of signal transduction requires the binding of regulatory molecules to their cognate targets. However, the crowded cell interior risks off-target interactions between proteins that are functionally unrelated. How such off-target interactions impact fitness is not generally known, but quantifying this is required to understand the constraints faced by cell systems as they evolve. Here, we use the model organism S. cerevisiae to inducibly express tyrosine kinases. Because yeast lacks bona fide tyrosine kinases, most of the resulting tyrosine phosphorylation is spurious. This provides a suitable system to measure the impact of artificial protein interactions on fitness. We engineered 44 yeast strains each expressing a tyrosine kinase, and quantitatively analysed their phosphoproteomes. This analysis resulted in ~30,000 phosphosites mapping to ~3,500 proteins. Examination of the fitness costs in each strain revealed a strong correlation between the number of spurious pY sites and decreased growth. Moreover, the analysis of pY effects on protein structure and on protein function revealed over 1000 pY events that we predict to be deleterious. However, we also find that a large number of the spurious pY sites have a negligible effect on fitness, possibly because of their low stoichiometry. This result is consistent with our evolutionary analyses demonstrating a lack of phosphotyrosine counter-selection in species with bona fide tyrosine kinases. Taken together, our results suggest that, alongside the risk for toxicity, the cell can tolerate a large degree of non-functional crosstalk as interaction networks evolve.

Unveiling the peptidome diversity of Lachesismuta snake venom: Discovery of novel fragments of metalloproteinase, l-amino acid oxidase, and bradykinin potentiating peptides.

Snake venoms are known to be major sources of peptides with different pharmacological properties. In this study, we comprehensively explored the venom peptidomes of three specimens of Lachesismuta, the largest venomous snake in South America, using mass spectrometry techniques. The analysis revealed 19 main chromatographic peaks common to all specimens. A total of 151 peptides were identified, including 69 from a metalloproteinase, 58 from the BPP-CNP precursor, and 24 from a l-amino acid oxidase. To our knowledge, 126 of these peptides were reported for the first time in this work, including a new SVMP-derived peptide fragment, Lm-10a. Our findings highlight the dynamic nature of toxin maturation in snake venoms, driven by proteolytic processing, post-translational modifications, and cryptide formation.

Long-term oncological and surgical outcomes after Video Endoscopic Inguinal Lymphadenectomy (VEIL) in patients with penile cancer

ABSTRACT Objective: To report outcomes from the largest multicenter series of penile cancer patients undergoing video endoscopic inguinal lymphadenectomy (VEIL). Materials and Methods: Retrospective multicenter analysis. Authors of 21 centers from the Penile Cancer Collaborative Coalition-Latin America (PeC-LA) were included. All centers performed the procedure following the same previously described standardized technique. Inclusion criteria included penile cancer patients with no palpable lymph nodes and intermediate/high-risk disease and those with non-fixed palpable lymph nodes less than 4 cm in diameter. Categorical variables are shown as percentages and frequencies whereas continuous variables as mean and range. Results: From 2006 to 2020, 210 VEIL procedures were performed in 105 patients. Mean age was 58 (45-68) years old. Mean operative time was 90 minutes (60-120). Mean lymph node yield was 10 nodes (6-16). Complication rate was 15.7%, including severe complications in 1.9% of procedures. Lymphatic and skin complications were noted in 8.6 and 4.8% of patients, respectively. Histopathological analysis revealed lymph node involvement in 26.7% of patients with non-palpable nodes. Inguinal recurrence was observed in 2.8% of patients. 10y- overall survival was 74.2% and 10-y cancer specific survival was 84.8%. CSS for pN0, pN1, pN2 and pN3 were 100%, 82.4%, 72.7% and 9.1%, respectively. Conclusion: VEIL seems to offer appropriate long term oncological control with minimal morbidity. In the absence of non-invasive stratification measures such as dynamic sentinel node biopsy, VEIL emerged as the alternative for the management of non-bulky lymph nodes in penile cancer.