Impact of the COVID-19 Pandemic on Cancer Diagnosis in Madrid (Spain) Based on the RTMAD Tumor Registry (2019-2021).

The coronavirus disease 2019 (COVID-19) pandemic has caused a significant disruption to cancer diagnosis, treatment and prevention worldwide that could have serious consequences in the near future. We intend to evaluate the weight of this backlog on a community-wide scale in Madrid during the period 2020-2021, and whether a stage shift towards the advanced stage has occurred. Cancer diagnoses in the Madrid tumor registry (RTMAD) from 2019-2021 were evaluated. Absolute and percentage differences in annual volume and observed-to-expected (O/E) volume ratios were calculated. Standardized incidence ratios (SIRs) and 95% confidence intervals (CIs) were calculated using the O/E ratio. The SIR for 2020-2021 compared to 2019 was 94.5% (95% CI 93.8-95.3), with unequal gender-specific cancer diagnosis recovery (88.5% for males and 102.1% for females). Most cancer types were underdiagnosed in 2020. The tendency worsened in 2021 for colorectal and prostate cancers (87.8%), but lung cancer recovered (102.1%) and breast cancer was over-diagnosed (114.4%) compared with reference pre-COVID-19 data. These changes have modified the ranking of the most frequent malignancies diagnosed in Madrid. Breast cancer has overtaken colorectal and prostate cancers, displaced to second and third position, respectively. Not only was colorectal cancer diagnosis affected more as a consequence of the COVID-19 pandemic but diagnosis of this malignancy at the advance stage also increased by 3.6% in 2020 and 4.2% in 2021 compared to the reference period of 2019. In summary, there is a large volume of undetected cancer in Madrid caused by the reduced access to care secondary to the COVID-19 pandemic, especially regarding colorectal and prostate cancer. Strategies are needed to recover the backlog of diagnoses and effectively treat these cases in the future and solve the negative impact that will be caused by the diagnostic delay. Analyzing the impact of new diagnoses suffered by each different malignancy and their recovery will help to understand how the future allocation of resources should look.

Hyperthermic Mitomycin C in Intermediate-risk Non-muscle-invasive Bladder Cancer: Results of the HIVEC-1 Trial.

BACKGROUND: Optimising therapeutic strategies of intermediate-risk non-muscle-invasive bladder cancer (IR-NMIBC) is needed. OBJECTIVE: To compare recurrence-free survival (RFS) with adjuvant intravesical mitomycin C (MMC) at normothermia or hyperthermia using the COMBAT bladder recirculation system at 43 °C for 30 and 60 min. DESIGN, SETTING, AND PARTICIPANTS: A prospective open-label, phase 3 randomised controlled trial (HIVEC-1) accrued across 13 centres between 2014 and 2020 in Spain. After complete transurethral resection of the bladder and immediate postoperative MMC instillation, patients with IR-NMIBC were randomised (1:1:1) to four weekly followed by three monthly 40-mg MMC instillations at normothermia (control; n = 106), 43 °C for 30 min (n = 107), or 43 °C for 60 min (n = 106) were investigated. Therapeutic compliance was defined as four or more instillations. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary outcome was RFS at 24 mo in the intention-to-treat (ITT) and per-protocol (PP) populations. The secondary outcomes included progression-free survival at 24 mo, safety outcome measures, and changes in health-related quality of life. Log-rank, Fisher, χ2, and analysis of variance tests were used. RESULTS AND LIMITATIONS: The ITT 24-mo RFS was 77% for control, 82% for 43 °C-30 min, and 80% for 43 °C-60 min (p = 0.6). The PP 24-mo RFS was 77% for control, 83% for 43 °C-30 min, and 80% for 43 °C-60 min (p = 0.59). Six patients progressed to muscle-invasive disease in the ITT population (four in the control, 43 °C-30 min, and 43 °C-60 min groups each) and four in the PP population (all controls). Serious adverse events occurred in 26 patients (8.1%), and we were unable to demonstrate a difference between groups (p = 0.5). Adverse events, mainly dysuria and spasms, occurred in 124 patients (33% in control, 35% in 43 °C-30 min, and 48% in 43 °C-60 min; p = 0.05). The total International Prostate Symptom Score worsened by 1.2 ±â€¯7.3 points, similarly across groups (p = 0.29). The Functional Assessment of Cancer Therapy-Bladder domains and indexes showed no significant change. CONCLUSIONS: Four-month adjuvant hyperthermic MMC using the COMBAT system for 30 and 60 min in IR-NMIBC is well tolerated, but we did not find it to be superior to normothermic MMC at 24 mo. PATIENT SUMMARY: We were unable to demonstrate the effectiveness of hyperthermia using the COMBAT system in intermediate-risk non-muscle-invasive bladder cancer. Further evaluation of long-term recurrence and progression, and maintenance regimens appears mandatory.

SEVA 4.0: an update of the Standard European Vector Architecture database for advanced analysis and programming of bacterial phenotypes.

The SEVA platform (https://seva-plasmids.com) was launched one decade ago, both as a database (DB) and as a physical repository of plasmid vectors for genetic analysis and engineering of Gram-negative bacteria with a structure and nomenclature that follows a strict, fixed architecture of functional DNA segments. While the current update keeps the basic features of earlier versions, the platform has been upgraded not only with many more ready-to-use plasmids but also with features that expand the range of target species, harmonize DNA assembly methods and enable new applications. In particular, SEVA 4.0 includes (i) a sub-collection of plasmids for easing the composition of multiple DNA segments with MoClo/Golden Gate technology, (ii) vectors for Gram-positive bacteria and yeast and [iii] off-the-shelf constructs with built-in functionalities. A growing collection of plasmids that capture part of the standard-but not its entirety-has been compiled also into the DB and repository as a separate corpus (SEVAsib) because of its value as a resource for constructing and deploying phenotypes of interest. Maintenance and curation of the DB were accompanied by dedicated diffusion and communication channels that make the SEVA platform a popular resource for genetic analyses, genome editing and bioengineering of a large number of microorganisms.

High-Efficiency Multi-site Genomic Editing (HEMSE) Made Easy.

The ability to engineer bacterial genomes in an efficient way is crucial for many bio-related technologies. Single-stranded (ss) DNA recombineering technology allows to introduce mutations within bacterial genomes in a very simple and straightforward way. This technology was initially developed for E. coli but was later extended to other organisms of interest, including the environmentally and metabolically versatile Pseudomonas putida. The technology is based on three pillars: (1) adoption of a phage recombinase that works effectively in the target strain, (2) ease of introduction of short ssDNA oligonucleotide that carries the mutation into the bacterial cells at stake and (3) momentary suppression of the endogenous mismatch repair (MMR) through transient expression of a dominant negative mutL allele. In this way, the recombinase protects the ssDNA and stimulates recombination, while MutLE36KPP temporarily inhibits the endogenous MMR system, thereby allowing the introduction of virtually any possible type of genomic edits. In this chapter, a protocol is detailed for easily performing recombineering experiments aimed at entering single and multiple changes in the chromosome of P. putida. This was made by implementing the workflow named High-Efficiency Multi-site genomic Editing (HEMSE), which delivers simultaneous mutations with a simple and effective protocol.

Functional mapping of PHF6 complexes in chromatin remodeling, replication dynamics, and DNA repair.

The Plant Homeodomain 6 gene (PHF6) encodes a nucleolar and chromatin-associated leukemia tumor suppressor with proposed roles in transcription regulation. However, specific molecular mechanisms controlled by PHF6 remain rudimentarily understood. Here we show that PHF6 engages multiple nucleosome remodeling protein complexes, including nucleosome remodeling and deacetylase, SWI/SNF and ISWI factors, the replication machinery and DNA repair proteins. Moreover, after DNA damage, PHF6 localizes to sites of DNA injury, and its loss impairs the resolution of DNA breaks, with consequent accumulation of single- and double-strand DNA lesions. Native chromatin immunoprecipitation sequencing analyses show that PHF6 specifically associates with difficult-to-replicate heterochromatin at satellite DNA regions enriched in histone H3 lysine 9 trimethyl marks, and single-molecule locus-specific analyses identify PHF6 as an important regulator of genomic stability at fragile sites. These results extend our understanding of the molecular mechanisms controlling hematopoietic stem cell homeostasis and leukemia transformation by placing PHF6 at the crossroads of chromatin remodeling, replicative fork dynamics, and DNA repair.

Propagation of Recombinant Genes through Complex Microbiomes with Synthetic Mini-RP4 Plasmid Vectors.

The promiscuous conjugation machinery of the Gram-negative plasmid RP4 has been reassembled in a minimized, highly transmissible vector for propagating genetically encoded traits through diverse types of naturally occurring microbial communities. To this end, the whole of the RP4-encoded transfer determinants (tra, mob genes, and origin of transfer oriT) was excised from their natural context, minimized, and recreated in the form of a streamlined DNA segment borne by an autoselective replicon. The resulting constructs (the pMATING series) could be self-transferred through a variety of prokaryotic and eukaryotic recipients employing such a rationally designed conjugal delivery device. Insertion of GFP reporter into pMATING exposed the value of this genetic tool for delivering heterologous genes to both specific mating partners and complex consortia (e.g., plant/soil rhizosphere). The results accredited the effective and functional transfer of the recombinant plasmids to a diversity of hosts. Yet the inspection of factors that limit interspecies DNA transfer in such scenarios uncovered type VI secretion systems as one of the factual barriers that check otherwise high conjugal frequencies of tested RP4 derivatives. We argue that the hereby presented programming of hyperpromiscuous gene transfer can become a phenomenal asset for the propagation of beneficial traits through various scales of the environmental microbiome.

Assessment of the Grades of Vesicoureteral Reflux in Stented Ureters: An Experimental Study.

INTRODUCTION: The aim of this experimental study is to assess, in a porcine model, the onset and grades of vesicoureteral reflux associated with ureteral stents. METHODS: Twenty-four female porcine models were used. A 4.7-Fr ureteral stent was placed in all right ureters and kept in place for 6 weeks. Follow-ups were performed on weeks 1, 3, 6, and 12. Ultrasonography, cystoscopy, and fluoroscopy were used to analyze grade of hydronephrosis, presence and grade of vesicoureteral reflux, bacteriuria, and macroscopic changes of the ureteral orifices. Vesicoureteral reflux was classified using a modification of the International Reflux Study Committee grades. RESULTS: 91.7% animals present vesicoureteral reflux, 89.5% grade IA, 3.5% grade IB, and 7% grade II. There is a significant increase in reflux during follow-ups at 3 and 6 weeks, whereas 6 weeks after removal, 26.3% of the ureters still present vesicoureteral reflux. Hydronephrosis and macroscopic changes of the ureteral orifice increase significantly with stenting, but there is no significant association between them and vesicoureteral reflux; the relationship between bacteriuria and the presence of vesicoureteral reflux is not significant either. CONCLUSION: Vesicoureteral reflux caused by ureteral stents in an animal model is mostly low grade and mainly affects the distal ureter.

Inhibition of DNA replication initiation by silver nanoclusters.

Silver nanoclusters (AgNCs) have outstanding physicochemical characteristics, including the ability to interact with proteins and DNA. Given the growing number of diagnostic and therapeutic applications of AgNCs, we evaluated the impact of AgNCs on DNA replication and DNA damage response in cell-free extracts prepared from unfertilized Xenopus laevis eggs. We find that, among a number of silver nanomaterials, AgNCs uniquely inhibited genomic DNA replication and abrogated the DNA replication checkpoint in cell-free extracts. AgNCs did not affect nuclear membrane or nucleosome assembly. AgNCs-supplemented extracts showed a strong defect in the loading of the mini chromosome maintenance (MCM) protein complex, the helicase that unwinds DNA ahead of replication forks. FLAG-AgNCs immunoprecipitation and mass spectrometry analysis of AgNCs associated proteins demonstrated direct interaction between MCM and AgNCs. Our studies indicate that AgNCs directly prevent the loading of MCM, blocking pre-replication complex (pre-RC) assembly and subsequent DNA replication initiation. Collectively, our findings broaden the scope of silver nanomaterials experimental applications, establishing AgNCs as a novel tool to study chromosomal DNA replication.

Refactoring the Conjugation Machinery of Promiscuous Plasmid RP4 into a Device for Conversion of Gram-Negative Isolates to Hfr Strains.

Chromosomal exchange and subsequent recombination of the cognate DNA between bacteria was one of the most useful genetic tools (e.g., Hfr strains) for genetic analyses of E. coli before the genomic era. In this paper, yeast assembly has been used to recruit the conjugation machinery of environmentally promiscuous RP4 plasmid into a minimized, synthetic construct that enables transfer of chromosomal segments between donor/recipient strains of P. putida KT2440 and potentially many other Gram-negative bacteria. The synthetic device features [i] a R6K suicidal plasmid backbone, [ii] a mini-Tn5 transposon vector, and [iii] the minimal set of genes necessary for active conjugation (RP4 Tra1 and Tra2 clusters) loaded as cargo in the mini-Tn5 mobile element. Upon insertion of the transposon in different genomic locations, the ability of P. putida-TRANS (transference of RP4-activated nucleotide segments) donor strains to mobilize genomic stretches of DNA into neighboring bacteria was tested. To this end, a P. putida double mutant ΔpyrF (uracil auxotroph) Δedd (unable to grow on glucose) was used as recipient in mating experiments, and the restoration of the pyrF+/edd+ phenotypes allowed for estimation of chromosomal transfer efficiency. Cells with the inserted transposon behaved in a manner similar to Hfr-like strains and were able to transfer up to 23% of their genome at frequencies close to 10-6 exconjugants per recipient cell. The hereby described TRANS device not only expands the molecular toolbox for P. putida, but it also enables a suite of genomic manipulations which were thus far only possible with domesticated laboratory strains and species.

The faulty SOS response of Pseudomonas putida KT2440 stems from an inefficient RecA-LexA interplay.

Despite its environmental robustness Pseudomonas putida strain KT2440 is very sensitive to DNA damage and displays poor homologous recombination efficiencies. To gain an insight into this deficiency isogenic ∆recA and ∆lexA1 derivatives of prophage-free strain P. putida EM173 were generated and responses of the recA and lexA1 promoters to DNA damage tested with GFP reporter technology. Basal expression of recA and lexA1 of P. putida were high in the absence of DNA damage and only moderately induced by norfloxacin. A similar behaviour was observed when equivalent GFP fusions to the recA and lexA promoters of E. coli were placed in P. putida EM173. In contrast, all SOS promoters were subject to strong repression in E. coli, which was released only when cells were treated with the antibiotic. Replacement of P. putida's native LexA1 and RecA by E. coli homologues did not improve the responsiveness of the indigenous functions to DNA damage. Taken together, it seems that P. putida fails to mount a strong SOS response due to the inefficacy of the crucial RecA-LexA interplay largely tractable to the weakness of the corresponding promoters and the inability of the repressor to shut them down entirely in the absence of DNA damage.

An automated DIY framework for experimental evolution of Pseudomonas putida.

Adaptive laboratory evolution (ALE) is a general and effective strategy for optimizing the design of engineered genetic circuits and upgrading metabolic phenotypes. However, the specific characteristics of each microorganism typically ask for exclusive conditions that need to be adjusted to the biological chassis at stake. In this work, we have adopted a do-it-yourself (DIY) approach to implement a flexible and automated framework for performing ALE experiments with the environmental bacterium and metabolic engineering platform Pseudomonas putida. The setup includes a dual-chamber semi-continuous log-phase bioreactor design combined with an anti-biofilm layout to manage specific traits of this bacterium in long-term cultivation experiments. As a way of validation, the prototype was instrumental for selecting fast-growing variants of a P. putida strain engineered to metabolize D-xylose as sole carbon and energy source after running an automated 42 days protocol of iterative regrowth. Several genomic changes were identified in the evolved population that pinpointed the role of RNA polymerase in controlling overall physiological conditions during metabolism of the new carbon source.

High-Efficiency Multi-site Genomic Editing of Pseudomonas putida through Thermoinducible ssDNA Recombineering.

Application of single-stranded DNA recombineering for genome editing of species other than enterobacteria is limited by the efficiency of the recombinase and the action of endogenous mismatch repair (MMR) systems. In this work we have set up a genetic system for entering multiple changes in the chromosome of the biotechnologically relevant strain EM42 of Pseudomononas putida. To this end high-level heat-inducible co-transcription of the rec2 recombinase and P. putida's allele mutLE36KPP was designed under the control of the PL/cI857 system. Cycles of short thermal shifts followed by transformation with a suite of mutagenic oligos delivered different types of genomic changes at frequencies up to 10% per single modification. The same approach was instrumental to super-diversify short chromosomal portions for creating libraries of functional genomic segments-e.g., ribosomal-binding sites. These results enabled multiplexing of genome engineering of P. putida, as required for metabolic reprogramming of this important synthetic biology chassis.

Multifunctional SEVA shuttle vectors for actinomycetes and Gram-negative bacteria.

Actinomycetales, such as the genus Streptomyces, are well-known cell factories employed to produce a wide variety of secondary metabolites for industrial use. However, not only is the genetic engineering of Streptomyces more complicated and tedious than other model laboratory species, such as Escherichia coli, there is also a considerable lack of genetic tools, hindering its adoption as a common chassis for synthetic biology. In this work, 23 novel shuttle vectors are presented that follow the canonical SEVA (Standard European Vector Architecture) common architecture with the goal of increasing the genetic toolbox repertoire for Streptomyces and other actinomycetes. The ORI module of these plasmids is composed of the combination of two origins of replication, one for Gram-negative bacteria and the other for Streptomyces, a Gram-positive bacteria. Origins of replication have been included in the collection for integrative, low-copy number, and medium-to-high-copy number vectors for Streptomyces. Also, a new selection marker has been developed that confers resistance to apramycin. The functionality of these plasmids was tested via the heterologous expression of GFP and the heterologous production of the plant flavonoid apigenin in Streptomyces albus J1074, with successful results in both cases, therefore expanding the current repertoire of genetic manipulation tools in Streptomyces species.

Ureteroiliac Fistula: Bleeding of Unknown Origin-Case Report and Review of the Literature.

Background: Ureteroiliac fistula is a rare cause of gross hematuria and lateralizing flank pain. Risk factors include previous pelvic surgery, pelvic radiotherapy, or chronic ureteral stentings. Diagnosis is challenging and requires arteriography and ureteroscopy. Management ranges from open surgery to minimally invasive means such as the use of an endovascular stent. Case Report: A 62-year-old man with postradical cystoprostatectomy and cutaneous ureterostomy presented an intermittent gross hematuria with anemia that required blood transfusions. Some CT arteriographies were performed but none of them could identify the bleeding origin. Therefore, a flexible ureteroscopy was performed that showed a left ureteroiliac fistula. Subsequently, an endovascular stent was placed in the left common iliac without complications. Conclusion: The ureteroiliac fistula is a life-threatening condition. CT arteriography or ureteroscopy might help in the diagnosis but the sensitivity is ∼64%. Arteriography with endovascular stenting is a viable and safe option. However, because of its rarity, long-term durable benefits still need to be documented.

Mismatch repair hierarchy of Pseudomonas putida revealed by mutagenic ssDNA recombineering of the pyrF gene.

The mismatch repair (MMR) system is one of the key molecular devices that prokaryotic cells have for ensuring fidelity of DNA replication. While the canonical MMR of E. coli involves 3 proteins (encoded by mutS, mutL and mutH), the soil bacterium Pseudomonads putida has only 2 bona fide homologues (mutS and mutL) and the sensitivity of this abridged system to different types of mismatches is unknown. In this background, sensitivity to MMR of this bacterium was inspected through single stranded (ss) DNA recombineering of the pyrF gene (the prokaryotic equivalent to yeast's URA3) with mutagenic oligos representative of every possible mispairing under either wild-type conditions, permanent deletion of mutS or transient loss of mutL activity (brought about by the thermoinducible dominant negative allele mutLE36K ). Analysis of single nucleotide mutations borne by clones resistant to fluoroorotic acid (5FOA, the target of wild type PyrF) pinpointed prohibited and tolerated single-nucleotide replacements and exposed a clear grading of mismatch recognition. The resulting data unequivocally established the hierarchy A:G < C:C < G:A < C:A, A:A, G:G, T:T, T:G, A:C, C:T < G:T, T:C as the one prevalent in Pseudomonas putida. This information is vital for enabling recombineering strategies aimed at single-nucleotide changes in this biotechnologically important species.

A Broad Host Range Plasmid-Based Roadmap for ssDNA-Based Recombineering in Gram-Negative Bacteria.

Recombineering is the use of phage recombination proteins to improve and facilitate bacterial genome engineering. Depending on the nature of the DNA template, double-stranded or single-stranded, the system needs three proteins (Gam, Exo, and Beta) or just one (Beta) to work properly. The use of this technique has been fundamental not only toward solving fundamental biological questions with reverse genetics but also for the generation of deep-engineered E. coli chassis strains. Unfortunately, the use of ssDNA recombineering is still limited to a narrow number of bacterial species. One of the reasons for that is the lack of proper recombinases to be efficiently used in different microorganisms and the lack of proper genetic tools to deliver and express this activity in a controlled way. Here, we describe a protocol to follow a simple workflow to identify, clone, and quantify the function of the selected recombinases in the organism of choice by cloning and expressing them in standardized broad host range plasmids. As an example of the method, we tested the use of the Ssr recombinase in P. putida EM42 by introducing a complete deletion of the target gene pyrF. The example shows how two parameters of the mutagenic oligo, i.e., length and phosphorothioate protection, affect the final outcome of the procedure.

[Video endoscopic inguinal lymphadenectomy in penile cancer: Systematic review.] / Linfadenectomía inguinal videoendoscópica en cáncer de pene: revisión sistemática.

OBJECTIVES: Penile cancer is not very frequent. To control the disease oncologically, we must perform inguinal lymphadenectomy in cases of high-risk histology, poor prognosis and palpable lymph nodes. The open inguinal lymphadenectomy has a high rate of morbidity. Consequently, this systematic review intends to summarize the published literature regarding the oncologic and post-surgery outcomes in video-endoscopic inguinal lymphadenectomy (VEIL). METHODS: A literature search has conducted through Pubmed, EMBASE and Cochrane library for English and Spanish articles. RESULTS: Our literature search identified 12 articles. In total, 161 patients have been subjected to 226 VEIL. Their average age was 55.66 years. In the case of open inguinal lymphadenectomy, 90 patients have been subjected to 106 operations. The rate of cutaneous complications was 6% for VEIL and 55.6% for open lymphadenectomy. The rate of lymphatic complications was very similar in both types of lymphadenectomy. The average number of lymph nodes obtained was 9.12 for VEIL and 7.02 lymph nodes for the open approach. CONCLUSION: Video-endoscopic inguinal lymphadenectomy contributes to less morbidity with a lower- rate of cutaneous complications and less severity. Furthermore, VEIL gives lower hospital stay without changing in initial oncologic outcomes. Although we need longer series to stablish the oncologic long-term results.

OBJETIVO: El cáncer de pene es una entidad poco frecuente. Para realizar un buen control oncológico, se recomienda la realización de linfadenectomía inguinal en casos de factores de riesgo de mal pronóstico, grado histológico alto o ganglios palpables o positivos. La linfadenectomía inguinal abierta presenta una alta tasa de morbilidad, por lo que en esta revisión se pretende resumir la literatura publicada en cuanto a los resultados oncológicos y postquirúrgicos en la linfadenectomía inguinal videoendoscópica (VEIL).MATERIAL Y MÉTODOS: Se realiza revisión sistemática de la literatura obtenida en "Pubmed", "EMBASE" y Cochrane library para artículos en inglés y español. RESULTADOS: Se han analizado un total de 12 artículos, que globalmente incluyen a 161 pacientes con 226 VEIL y una edad media de 55,66 años y 90 pacientes a los que se les ha realizado 106 linfadenectomías abiertas. En el caso del VEIL se han presentado 6% de complicaciones cutáneas y del 55,6% en el caso de la vía abierta. En cuanto a las complicaciones linfáticas, no hay diferencias significativas. La media de ganglios extraídos en el caso de VEIL de 9,12 ganglios y de 7,09 ganglios en abordaje abierto. CCONCLUSIONES: La linfadenectomía inguinal videoendoscópica aporta una menor morbilidad, con una menor tasa de complicaciones cutáneas, y de menor gravedad. Asimismo, aporta menor estancia hospitalaria, sin afectación de los resultados oncológicos iniciales. Aunque se necesitan series con mayor tiempo de seguimiento para valoración de resultados oncológicos a largo plazo.

Linfadenectomía inguinal videoendoscópica en cáncer de pene: revisión sistemática / Video endoscopic inguinal lymphadenectomy in penile cancer: Systematic review

OBJETIVO: El cáncer de pene es una entidad poco frecuente. Para realizar un buen control oncológico, se recomienda la realización de linfadenectomía inguinal en casos de factores de riesgo de mal pronóstico, grado histológico alto o ganglios palpables o positivos. La linfadenectomía inguinal abierta presenta una alta tasa de morbilidad, por lo que en esta revisión se pretende resumir la literatura publicada en cuanto a los resultados oncológicos y postquirúrgicos en la linfadenectomía inguinal videoendoscópica (VEIL). MATERIAL Y MÉTODOS: Se realiza revisión sistemática de la literatura obtenida en "Pubmed", "EMBASE" y Cochrane library para artículos en inglés y español. RESULTADOS: Se han analizado un total de 12 artículos, que globalmente incluyen a 161 pacientes con 226 VEIL y una edad media de 55,66 años y 90 pacientes a los que se les ha realizado 106 linfadenectomías abiertas. En el caso del VEIL se han presentado 6% de complicaciones cutáneas y del 55,6% en el caso de la vía abierta. En cuanto a las complicaciones linfáticas, no hay diferencias significativas. La media de ganglios extraídos en el caso de VEIL de 9,12 ganglios y de 7,09 ganglios en abordaje abierto. CONCLUSIONES: La linfadenectomía inguinal videoendoscópica aporta una menor morbilidad, con una menor tasa de complicaciones cutáneas, y de menor gravedad. Asimismo, aporta menor estancia hospitalaria, sin afectación de los resultados oncológicos iniciales. Aunque se necesitan series con mayor tiempo de seguimiento para valoración de resultados oncológicos a largo plazo

OBJECTIVES: Penile cancer is not very frequent. To control the disease oncologically, we must perform inguinal lymphadenectomy in cases of high-risk histology, poor prognosis and palpable lymph nodes. The open inguinal lymphadenectomy has a high rate of morbidity. Consequently, this systematic review intends to summarize the published literature regarding the oncologic and post-surgery outcomes METHODS: A literature search has conducted through Pubmed, EMBASE and Cochrane library for English and Spanish articles. RESULTS: Our literature search identified 12 articles. In total, 161 patients have been subjected to 226 VEIL. Their average age was 55.66 years. In the case of open inguinal lymphadenectomy, 90 patients have been subjected to 106 operations. The rate of cutaneous complications was 6% for VEIL and 55.6% for open lymphadenectomy. The rate of lymphatic complications was very similar in both types of lymphadenectomy. The average number of lymph nodes obtained was 9.12 for VEIL and 7.02 lymph nodes for the open approach. CONCLUSION: Video-endoscopic inguinal lymphadenectomy contributes to less morbidity with a lower-rate of cutaneous complications and less severity. Furthermore, VEIL gives lower hospital stay without changing in initial oncologic outcomes. Although we need longer series to stablish the oncologic long-term results

CRISPR/Cas9-enhanced ssDNA recombineering for Pseudomonas putida.

Implementation of single-stranded DNA (ssDNA) recombineering in Pseudomonas putida has widened the range of genetic manipulations applicable to this biotechnologically relevant bacterium. Yet, the relatively low efficiency of the technology hampers identification of mutated clones lacking conspicuous phenotypes. Fortunately, the use of CRISPR/Cas9 as a device for counterselection of wild-type sequences helps to overcome this limitation. Merging ssDNA recombineering with CRISPR/Cas9 thus enables a suite of genomic edits with a straightforward approach: a CRISPR plasmid provides the spacer DNA sequence that directs the Cas9 nuclease ribonucleoprotein complex to cleave the genome at the wild-type sequences that have not undergone the change entered by the mutagenic ssDNA oligonucleotide(s). This protocol describes a complete workflow of the method optimized for P. putida, although it could in principle be applicable to many other pseudomonads. As an example, we show the deletion of the edd gene that encodes one key enzyme that operates the EDEMP cycle for glucose metabolism in P. putida EM42. By combining two incompatible CRISPR plasmids with different antibiotic selection markers, we show that the procedure can be cycled to implement consecutive deletions in the same strain, e.g. deletion of the pyrF gene following that of the edd mutant. This approach adds to the wealth of genetic technologies available for P. putida and strengthens its status as a chassis of choice for a suite of biotechnological applications.

Improved Thermotolerance of Genome-Reduced Pseudomonas putida EM42 Enables Effective Functioning of the PL /cI857 System.

The higher intracellular ATP levels of genome-edited strains of P. putida that result from deleting various energy-consuming functions has been exploited for expanding the window of thermal tolerance of this bacterium. Unlike instant growth halt and eventual death of the naturally occurring strain P. putida KT2440 at 42 °C, the EM42 variant maintained growth and viability of most of the population at the higher temperature for at least 6 h. The authors took advantage of this quality for implementing a robust thermo-inducible heterologous expression device in this species. To this end, the cI857/PL pair of the lambda phage of Escherichia coli was reshaped as a functional cargo that followed the SEVA (Standard European Vector Architecture) format. Quantitation of the transcriptional output of the resulting expression device with GFP reporter technology in various gene dosages identified conditions of unprecedented induced/uninduced ratios (>300 folds) and very high total transcriptional capacity in this bacterial host. The broad-host range nature of the cognate replication origins makes expression vectors pSEVA2214 (low plasmid copy number), pSEVA2314 (medium), and pSEVA2514 (high) to cover a wide range of heterologous expression needs in P. putida and possibly other Gram-negative species.